METHOD FOR ENCODING DIGITAL DATA ON NUCLEIC ACIDS USING

BIOLOGICAL PROCESSES

FIELD OF INVENTION

[0001] The present invention relates to nucleic acid-based data storage methods for storing digital information.

BACKGROUND OF INVENTION

[0002] Storing and archiving digital data are major issues in our modem societies. The current digital media stored in data centers are fragile, bulky and energy-consuming. Although optical media, magnetic tapes, hard drives or flash memory have been developed, their durability does not exceed ten years on average. These data must be regularly copied onto new reliable media and have to be maintained at controlled temperature and humidity, inducing a colossal energy cost and requiring huge amounts of raw materials. The amount of energy consumed by data centers corresponds to 2% of the worldwide electricity consumption (Masanet et al. 2020). The carbon footprint of the data centers exceeds that of global civil aviation. Despite their energy cost, their carbon footprint and their increasing need for bulky area, data centers can only store 30% of the data we produce while our data production grows exponentially: “If today we are capable of storing about 30% of the information we generate, in only 10 or 12 years we will be able to store about 3%” (Dr. Karin Strauss, Microsoft Research, 2018). Given these general considerations, the data revolution, the big data market and the development of artificial intelligence cannot be pursued without finding innovative solutions to the problem of data storage.

[0003] US2018/0137418 describes the use of chemically produced DNA bricks and assembles several of them (3-6) to make a larger molecule (a few hundred base pairs) to encode the information bit (0 or 1). However, these processes are time consuming and costly. [0004] Consequently, there is still a need for new means for storing digital data that can sustain encoding of large amounts of data, and can further be biocompatible, i.e., that can be copied, edited, written and/or read using living organisms.

SUMMARY

[0005] The present invention relates to a nucleic acid-based data storage method for storing information comprising: a) recovering data in the form of a digital sequence formed of a plurality of bits, each bit having the value 0 or 1 , b) subdividing the digital sequence into n digital subsequences, each comprising m bits, m being comprised between 2 and 16, c) converting each of the n digital subsequences into a bioblock, a bioblock consisting of a sequence of m nucleotides, wherein the digital subsequence consists in m bits assigned to positions 0 to m- 1, and wherein the conversion of a digital subsequence into a bioblock consists in: converting bits at even positions to a first nucleotide N 1 if said bits has the value 0, and to a second distinct nucleotide N2 if said bits has the value 1 and converting bits at odd positions to a third nucleotide N3 if said bits has the value 0, and to a fourth distinct nucleotide N4 if said bits has the value 1, wherein N 1 , N2, N3 and N4 are distinct nucleotides d) constructing a plurality of x components, each individual component of the plurality of x components comprising at least one bioblock, and the x components together comprising n bioblocks e) assembling together in a fixed order, in one or more steps, the plurality of x components.

[0006] In some embodiments, the nucleotides are selected from the group of natural nucleotides consisting of adenine, guanine, cytosine, uracil and thymine or from nonnatural nucleotides. [0007] In some embodiments, the x components are x DNA molecules, preferably x double-stranded DNA molecules.

[0008] In some embodiments, at step (d) the construction of a plurality of x components, each comprising at least one bioblock, comprises the steps of: selectively capturing x data storage nucleic acid molecules from at least one library of data storage nucleic acid molecules, wherein each data storage nucleic acid molecule comprises at least one bioblock surrounded by regions comprising cleavage sites, cleaving each of the x data storage nucleic acid molecules, thereby releasing the at least one bioblock.

[0009] In some embodiments, at step (d) the construction of a plurality of x components, each comprising at least one bioblock, comprises the steps of: selectively capturing n data storage nucleic acid molecules from at least two libraries of data storage nucleic acid molecules, wherein each data storage nucleic acid molecule of each library comprises one bioblock surrounded by regions comprising cleavage sites, and wherein each library comprises all possible bioblocks of m nucleotides, cleaving each of the n data storage nucleic acid molecules, thereby releasing the n bioblocks.

[0010] In some embodiments, the regions comprising cleavage sites comprises from 2 to 25 nucleotides.

[0011] In some embodiments, the region surrounding each bioblock comprises a site for a restriction enzyme, and step (d) comprises a step of digesting each of the x data storage nucleic acid molecules with one or two restriction enzymes.

[0012] In some embodiments, step (e) comprises one or several assembling steps using overlap-extension polymerase chain reaction (PCR), polymerase cycling assembly, sticky end ligation, biobricks assembly, golden gate assembly, Gibson assembly, recombinase assembly, ligase cycling reaction, template directed ligation, in vivo assembly or any other DNA assembly protocol.

[0013] The present invention further relates to a data storage nucleic acid molecule comprising at least one bioblock, a bioblock consisting of a nucleic acid sequence consisting of m nucleotides assigned to positions 0 to m-1, wherein a bioblock is formed of at least 2 and at most 4 distinct nucleotides

- nucleotides at even positions may be selected from a first and a second nucleotide, and nucleotides at odd positions may be selected from a third and a fourth nucleotide, said first, second, third and fourth nucleotides being distinct.

[0014] In some embodiments, the data storage nucleic acid molecule is a doublestranded molecule, preferably a DNA molecule.

[0015] In some embodiments, the data storage nucleic acid molecule is a plasmid, a cosmid, a fosmid, a prokaryotic chromosome or a eukaryotic chromosome.

[0016] In some embodiments, each of the bioblock is surrounded by regions comprising cleavage sites, preferably by two sites for one restriction enzyme.

[0017] In some embodiments, the data storage nucleic acid molecule is replicative.

[0018] The present invention further relates to a library comprising a plurality of data storage nucleic acid molecules according to the invention, wherein each of the data storage nucleic acid molecule of the library contains one bioblock, wherein each data storage nucleic acid molecule of the library comprises the same surrounding regions comprising cleavage sites and wherein the library contains all possible bioblocks of m nucleotides.

[0019] The present invention further relates to a nucleic acid-based data storage system comprising at least two libraries according to the invention. DEFINITIONS

[0020] In the present invention, the following terms have the following meanings:

[0021] The term “digital data” refers to data that can be managed by computerized machines. As used herein, the expression “digital data” is meant to refer to data represented by a binary system. As used herein, a “binary system” refers to a language composed of bits “0” and “1”. Non-limitative examples of digital data may be program files, text files, music files, image files, video files and combinations thereof.

[0022] The term “storage” or “storing” refers to the action of keeping an item in a specific place for future use or for safekeeping. More specifically, the expression “storage of digital data” is intended to mean the action of safely keeping the digital information for further use.

[0023] The term “replicative” refers to the ability to be replicated in vivo by a polymerase, such as, e.g., a DNA polymerase, i.e., to be exactly duplicated, within the margin of error of replication mechanisms of living organisms. As used herein, a “replicative nucleic acid molecule” is intended to refer to a nucleic acid molecule that can be copied at least once in vivo. In one embodiment, the nucleic acid molecule according to the invention is selected in the group consisting of a plasmid, a cosmid and a chromosome. In practice, a replicative nucleic acid molecule comprises one or more origin(s) of replication (also termed ORI), or one or more centromere(s) (for chromosomes).

[0024] Within the scope of the present invention, the term “nucleotide” and “nucleic base” are meant as substitutes for one another and are intended to refer to the nucleic building block of a DNA or RNA molecule. Nucleotides comprise both natural nucleotides and non-natural nucleotides. As used herein, a natural nucleotide refers to a purine Adenine (A) or Guanine (G); or to a pyrimidine Cytosine (C), Thymine (T) or Uracil (U). For DNA nucleic acids, A refers to the dAMP deoxyribonucleotide; G refers to the dGMP deoxyribonucleotide; C refers to the dCMP deoxyribonucleotide; and T refers to the dTMP deoxyribonucleotide. For RNA nucleic acids, A refers to the AMP ribonucleotide; G refers to the GMP ribonucleotide; C refers to the CMP ribonucleotide; and U refers to the UMP ribonucleotide. As used herein, the term “non-natural nucleotides” refers to chemically modified A, T, U, C or G nucleotides. Non limitative examples of non-natural nucleotides include 2-Amino-ATP, 8-Aza-ATP, 2'-Fluoro- dATP, 2'-Fluoro-dCTP, 2’-Fluoro-dGTP, 2’-Fluoro-dUTP, 5-Iodo-CTP, 5-Iodo-UTP, N6-Methyl-ATP, 5-Methyl-CTP, 2’-0-Methyl-ATP, 2’-0-Methyl-CTP, 2’-0-Methyl-GTP, 2'-0-Methyl-UTP, Pseudo-UTP, ITP, 2'-0-Methyl-ITP, Puromycin-TP, Xanthosine -TP, 5-Methyl-UTP, 4-Thio-UTP, 2’-Amino-dCTP, 2’-Amino-dUTP, 2’-Azido-dCTP, 2'- Azido-dUTP, 06-Methyl-GTP, 2-Thio-UTP, Ara-CTP, Ara-UTP, 5,6-Dihydro-UTP, 2- Thio-CTP, 6-Aza-CTP, 6-Aza-UTP, Nl-Methyl-GTP, 2’-0-Methyl-2-Amino-ATP, 2'-O- Methylpseudo-UTP, N1 -Methyl- ATP, 2’-O-Methyl-5-methyl-UTP, 7-Deaza-GTP, 2'- Azido-dATP, 2'-Amino-dATP, Ara-ATP, 8-Azido-ATP, 5-Bromo-CTP, 5-Bromo-UTP, 2'-Fluoro-dTTP, 3 '-O-Methyl- ATP, 3'-O-Methyl-CTP, 3'-O-Methyl-GTP, 3'-O-Methyl- UTP, 7-Deaza-ATP, 5-AA-UTP, 2'-Azido-dGTP, 2'-Amino-dGTP, 5-AA-CTP, 8-Oxo- GTP, Pseudoiso-CTP, N4-Methyl-CTP, N1 -Methylpseudo-UTP, 5,6-Dihydro-5- Methyl-UTP, N6-Methyl-Amino-ATP, 5-Carboxy-CTP, 5-Formyl-CTP, 5- Hydroxymethyl-UTP, 5-Hydroxymethyl-CTP, Thieno-GTP, 5-Hydroxy-CTP, 5-Formyl- UTP, Thieno-UTP, 2-Amino-dATP, 5-Bromo-dCTP, 5-Bromo-dUTP, 7-Deaza-dATP, 7- Deaza-dGTP, diTP, 5-Propynyl-dCTP, 5 -Propynyl-dUTP, 2'-dUTP, 5-Fluoro-dUTP, 5- lodo-dCTP, 5-Iodo-dUTP, N6-Methyl-dATP, 5-Methyl-dCTP, O6-Methyl-dGTP, N2- Methyl-dGTP, 8-Oxo-dATP, 8-Oxo-dGTP, 2-Thio-dTTP, 2'-dPTP, 5-Hydroxy-dCTP, 4- Thio-dTTP, 2-Thio-dCTP, 6-Aza-dUTP, 6-Thio-dGTP, 8-Chloro-dATP, 5-AA-dCTP, 5- AA-dUTP, N4-Methyl-dCTP, 2’-deoxyzebularine-TP, 5-Hydroxymethyl-dUTP, 5- Hydroxymethyl-dCTP, 5-Propargylamino-dCTP, 5-Propargylamino-dUTP, 5-Carboxy- dCTP, 5-Formyl-dCTP, 5-Indolyl- AA-dUTP, 5-Carboxy-dUTP, 5-Formyl-dUTP, 3'- dATP, 3'-dGTP, 3'-dCTP, 5-Methyl-3'-dUTP, 3'-dUTP, ddATP, ddGTP, ddUTP, ddTTP, ddCTP, 3'-Azido-ddATP, 3'-Azido-ddGTP, 3'-Azido-ddTTP, 3 '- Amino-ddATP, 3'- Amino-ddCTP, 3'-Amino-ddGTP, 3 '-Amino-ddTTP, 3'-Azido-ddCTP, 3'-Azido-ddUTP, 5-Bromo-ddUTP, ddITP, (l-Thio)-dATP, (l-Thio)-dCTP, (l-Thio)-dGTP, (1-Thio)- dTTP, (l-Thio)-ATP, (l-Thio)-CTP, (l-Thio)-GTP, (l-Thio)-UTP, (l-Thio)-ddATP, (1- Thio)-ddCTP, (l-Thio)-ddGTP, (l-Thio)-ddTTP, (l-Thio)-3'-Azido-ddTTP, (1-Thio)- ddUTP, (l-Borano)-dATP, (l-Borano)-dCTP, (l-Borano)-dGTP, (l-Borano)-dTTP, Ganciclovir- TP, Cidofovir-DP, 3-methyl-6-amino-5-(r-b-D-2'-deoxyribofuranosyl)- pyrimidin-2-one, 6-amino-9[(r-b-D-2'-deoxyribofuranosyl)-4-hydroxy-5-

(hydroxymethyl)-oxolan-2-yl]-lH-purin-2-one, 6-amino-3-(T-b-D-2'- deoxyribofuranosyl)-5 -nitro- lH-pyridin-2-one and 2-amino-8-(r-b-D-2'- deoxyribofuranosyl)-imidazo-[ 1 ,2a]- 1 ,3,5-triazin-[8H]-4-one.

DETAILED DESCRIPTION

[0025] The present invention relates to a nucleic acid-based data storage method for storing information comprising:

(a) recovering data in the form of a digital sequence formed of a plurality of bits, each bit having the value 0 or 1 ,

(b) subdividing the digital sequence into n digital subsequences, each comprising m bits, m being comprised between 2 and 16,

(c) converting each of the n digital subsequences into a bioblock, a bioblock consisting of a sequence of m nucleotides, wherein the digital subsequence consists in m bits assigned to positions 0 to m-1, and wherein the conversion of a digital subsequence into a bioblock consists in: converting bits at even positions to a first nucleotide N 1 if said bits has the value 0, and to a second distinct nucleotide N2 if said bits has the value 1 and converting bits at odd positions to a third nucleotide N3 if said bits has the value 0, and to a fourth distinct nucleotide N4 if said bits has the value 1, wherein N 1 , N2, N3 and N4 are distinct nucleotides

(d) constructing a plurality of x components, each individual component of the plurality of x components comprising at least one bioblock, and the x components together comprising n bioblocks (e) assembling together in a fixed order, in one or more steps, the plurality of x components.

[0026] As used herein, the term “bit” (binary digit) refers to the smallest base unit of digital information. In practice, a bit relies on a base-2 numeral system and can have the value of either 0 or 1. Methods to store bits involve the use of electronic devices and are well known in the art.

[0027] Within the scope of the present invention, the term “byte”, interchangeable with the terms “bit string” or “bit chain”, refers to a contiguous sequence of bits, herein also referred to as a “digital subsequence”. Within the scope of the present invention, the number of bits per byte corresponds to the value of m.

[0028] In one embodiment, the value of m is comprised between 2 and 16. As used herein, the term “between 2 and 16” means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16. In one embodiment, the value of m is selected from the group comprising or consisting of 2, 4, 6, 8, 10, 12, 14 and 16. In one embodiment, the value of m is selected from the group comprising or consisting of 2, 4, 8 and 16.

[0029] In one embodiment, the value of m is 8. In practice, a byte consisting of 8 bits is referred herein as an octet; and a bioblock resulting from the conversion of an octet is referred herein as a biooctet.

[0030] In one embodiment, the value of m is 16. In one embodiment, the value of m is 4. In one embodiment, the value of m is 2.

[0031] In one embodiment, the digital sequence may be comprised in, or consist of, any digital file stored on a computer. In one embodiment, the file is a file type selected from the group comprising ,3dm (Rhino 3D Model), ,3ds (3D Studio Scene), ,3g2 (3GPP2 multimedia file), ,3gp (3GPP multimedia File), .accdb (Access 2007 Database file), .ai (Adobe Illustrator file), .aif (AIF/Audio Interchange audio file), .apk (Android package file), .asp and .aspx (Active Server Page file), .avi (Audio Video Interleave file), .bak (Backup file), .bat (Batch file), .bin (Binary file), .bmp (Bitmap image file), .cab (Windows Cabinet file), .cda (CD audio track file), .cer (Internet security certificate), .cfg (Configuration file), .cfin (ColdFusion Markup file), .cgi (Common Gateway Interface Script), .cgi or .pl (Perl script file), .com (MS-DOS command file), .cpl (Windows Control panel file), .css (Cascading Style Sheet file), .csv (Comma separated value file), .cur (Windows cursor file), .dat (Data file), .db or .dbf (Database file), .dll (DLL file), .dmp (Dump file), .doc and .docx (Microsoft Word file), .drv (Device driver file), .exe (Executable file), .flv (Adobe Flash Video file), .gif (GIF/Graphical Interchange Format image), ,h264 (H.264 video file), .htm and .html (HTME/Hypertext Markup Eanguage file), .icns (macOS X icon resource file), .ico (Icon file), .ico (Icon file), .iff (Interchange File Format), .ini (Initialization file), .jar (Java Archive file), .jpeg or .jpg (JPEG image), .js (JavaScript file), .jsp (Java Server Page file), .key (Keynote presentation), .Ink (Windows shortcut file), .log (Fog file), ,m4v (Apple MP4 video file), .max (3ds Max Scene file), .mdb (Microsoft Access database file), .mid or .midi (MIDI audio file), .mkv (Matroska Multimedia Container), .mov (Apple QuickTime movie file), .mp3 (MP3 audio file), .mp4 (MPEG-4 Video File), .mpa (MPEG-2 audio file), .mpg or .mpeg (MPEG video file), .msg (Outlook Mail Message), .msi (Windows installer package), .obj (Wavefront 3D Object file), .odp (OpenOffice Impress presentation file), .ods (OpenOffice Calc spreadsheet file), .odt (OpenOffice Writer document file), .part (Partially downloaded file), .pdb (Program Database), .pdf (PDF file), .php (PHP Source Code file), .png (PNG / Portable Network Graphic image), .pps (PowerPoint slide show), .ppt (PowerPoint presentation), .pptx (PowerPoint Open XME presentation), .ps (PostScript file), .psd (PSD / Adobe Photoshop Document image), .py (Python file), .rm (Real Media file), .rss (RSS/Rich Site Summary file), .rtf (Rich Text Format file), .sav (Save file), .sql (SQE/Structured Query Eanguage database file), .svg (Scalable Vector Graphics file), .swf (Small Web Format file, formerly ShockWave Flash file), .sys (Windows system file), .tar (Einux / Unix tarball file archive), .tex (TeX document file), .tif or .tiff (TIFF image), .tmp (Temporary file), .txt (Plain text file), .vob (DVD Video Object file), .wav (WAVE file), .wks and .wps (Microsoft Works Word Processor Document file), .wma (Windows Media audio file), .wmv (Windows Media Video file), .wpd (WordPerfect document), .wpl (Windows Media Player playlist), .wsf (Windows Script File), .xhtml (XHTME / Extensible Hypertext Markup Eanguage file), .xlr (Microsoft Works spreadsheet file), .xls (Microsoft Excel file), .xlsx (Microsoft Excel Open XME spreadsheet file). [0032] In one embodiment, the digital sequence may be selected in a group comprising program files, text files, table files, audio files, image files, video files and combinations thereof.

[0033] In one embodiment, the digital sequence may be comprised in, or consist of, program files. Non-limitative examples of program files include .accdb (Access 2007 Database File), .apk (Android package file), .bak (Backup file), .bat (Batch file), .bin (Binary file), .cab (Windows Cabinet file), .cfg (Configuration file), .cgi (Common Gateway Interface Script), .com (MS-DOS command file), .cpl (Windows Control panel file), .csv (Comma separated value file), .cur (Windows cursor file), .dat (Data file), .db or .dbf (Database file), .dll (DLL file), .dmp (Dump file), .drv (Device driver file), .exe (Executable file), .icns (macOS X icon resource file), .ico (Icon file), .ini (Initialization file), .jar (Java Archive file), .Ink (Windows shortcut file), .log (Log file), .mdb (Microsoft Access database file), .msi (Windows installer package), .pdb (Program Database), .py (Python file), .sav (Save file), .sql (SQL/Structured Query Language database file), .sys (Windows system file), .tar (Linux / Unix tarball file archive), .tmp (Temporary file) and .wsf (Windows Script File).

[0034] In one embodiment, the digital sequence may be comprised in, or consist of, text files. Non-limitative examples of text files include .doc and .docx (Microsoft Word file), .odt (OpenOffice Writer document file), .msg (Outlook Mail Message), .pdf (PDF file), .rtf (Rich Text Format file), .tex (TeX document file), .txt (Plain text file), .wks and .wps (Microsoft Works Word Processor Document file), and .wpd (WordPerfect document).

[0035] In one embodiment, the digital sequence may be comprised in, or consist of, table files, e.g., spreadsheets. Non-limitative examples of table files include .ods (OpenOffice Calc spreadsheet file), .xlr (Microsoft Works spreadsheet file), .xls (Microsoft Excel file) and .xlsx (Microsoft Excel Open XME spreadsheet file).

[0036] In one embodiment, the digital sequence may be comprised in, or consist of, audio files, e.g., music files. Non-limitative examples of audio files include .aif (AIF/Audio Interchange audio file), .cda (CD audio track file), .iff (Interchange File Format), .mid or .midi (MIDI audio file), .mp3 (MP3 audio file), .mpa (MPEG-2 audio file), .wav (WAVE file), .wma (Windows Media audio file), and .wpl (Windows Media Player playlist).

[0037] In one embodiment, the digital sequence may be comprised in, or consist of, image files. Non-limitative examples of image files include .ai (Adobe Illustrator file), .bmp (Bitmap image file), .gif (GIF/Graphical Interchange Format image), .ico (Icon file), .jpeg or .jpg (JPEG image), .max (3ds Max Scene file), .obj (Wavefront 3D Object file), .png (PNG / Portable Network Graphic image), .ps (PostScript file), .eps (Encapsulated PostScript file), .psd (PSD / Adobe Photoshop Document image), .svg (Scalable Vector Graphics file), .tif or .tiff (TIFF image), ,3ds (3D Studio Scene), and ,3dm (Rhino 3D Model).

[0038] In one embodiment, the digital sequence may be comprised in, or consist of, video files. Non-limitative examples of video files include .avi (Audio Video Interleave File), .flv (Adobe Flash Video File), ,h264 (H.264 video File), ,m4v (Apple MP4 video File), .mkv (Matroska Multimedia Container), .mov (Apple QuickTime movie File), .mp4 (MPEG-4 Video File), .mpg or .mpeg (MPEG video File), .rm (Real Media File), .swf (Shockwave flash File), .vob (DVD Video Object File), .wmv (Windows Media Video File), ,3g2 (3GPP2 Multimedia File), and ,3gp (3GPP multimedia File).

[0039] In one embodiment, the total number of bytes, i.e., digital subsequences comprising m bits, in the digital sequence is termed n, wherein the value of n is at least one. As used herein, the term “at least one” encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 128, 256, 500, 512, 1000, 1024, 2048, 4096, 8192, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , 10 ¹⁶ , 10 ¹⁷ , 10 ¹⁸ , 10 ¹⁹ , 10 ² °, 10 ²¹ bytes, or more. Thus, in practice, the number of bits comprised in the digital sequence equals m (i.e., the number of bits per byte) multiplied by n i.e., the number of bytes, or digital subsequences, comprised in the digital sequence).

[0040] Each bit has a defined position within the digital subsequence (or byte) comprising m bits, the first position being position 0, the last position being equal to m- 1. Thus, the position of each bit in the digital sequence can be even or odd; wherein even positions comprise 0, 2, 4, 6, 8, 10, 12 and 14; and wherein odd positions comprise 1, 3, 5, 7, 9, 11, 13 and 15. In one embodiment, the digital subsequence is an octet; and even positions comprise 0, 2, 4 and 6, and odd positions comprise 1, 3, 5 and 7.

[0041] In one embodiment, the present invention comprises a step of converting a byte stored on an electronic device, into a byte stored on a nucleic acid molecule, wherein a byte stored on a nucleic acid molecule is herein referred to as a bioblock, and wherein a bioblock consists of m nucleotides. In one embodiment, the byte is an octet, i.e., m=8, and a bioblock is herein referred to as a biooctet.

[0042] In one embodiment, the bioblock comprises 2, 3 or 4 distinct nucleotides, wherein the distinct nucleotides are herein referred to as Nl, N2, N3 and N4. In one embodiment, a biooctet comprises exactly 4 distinct nucleotides.

[0043] In one embodiment, both the value and position of each bit comprised in the byte is encoded in the corresponding bioblock, wherein: bits having the value 0 and localized at even positions correspond to a first nucleotide N 1 ,

- bits having the value 1 and localized at even positions correspond to a second nucleotide N2,

- bits having the value 0 and localized at odd positions correspond to a third nucleotide N3, bits having the value 1 and localized at odd positions correspond to a fourth nucleotide N4, and wherein Nl, N2, N3 and N4 are distinct nucleotides.

[0044] The method according to the invention comprises constructing at least one component, preferably more than one component, wherein each component comprises or consists of at least one bioblock e.g., at least one biooctet), and wherein the total number of components is x. In one embodiment, the number of bioblocks (e.g., biooctet), per component is y, wherein the value of y is at least 1. In one embodiment, the value of x is n divided by y ( x = - ). [0045] As used herein, the term “more than one” means 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000 or more. As used herein, the term “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 128, 256, 500, 512, 1000, 10 ⁴ , 10 ⁵ , 10 ⁶ or more.

[0046] In one embodiment, each component comprises the same number of bioblocks. In one embodiment, x = n, i.e., y=l.

[0047] In another embodiment, x and n are distinct, i.e., y^l, meaning that each component comprises from 2 to n bioblocks (e.g., from 2 to n biooctet).

[0048] In certain embodiments, the value of x is not n divided by y.

[0049] In one embodiment, y does not have a fixed value, i.e., at least 2, 3, 4, 5 or more components comprise a distinct number of bioblocks. In certain embodiments, each component comprises a distinct number of bioblocks.

[0050] In certain embodiments, each component comprises the same number of bioblocks (y), except for one component that comprises from 1 to y-1 bioblocks, wherein the value of y is at least 2.

[0051] In one embodiment, the x components are assembled together in a fixed order, wherein the fixed order used for assembling the x components is identical to the order of the n digital subsequences within the digital sequence.

[0052] In one embodiment, the assembly of the x components is performed in one or more steps. In one embodiment, the assembly of the x components is performed in one step. In one embodiment, the assembly of the x components is performed in more than one step. In one embodiment, the assembly of the x components is performed sequentially, separately, simultaneously, or combinations thereof.

[0053] In one embodiment, the nucleotides are selected from the group consisting of natural nucleotides and non-natural nucleotides. [0054] Natural nucleotides include adenine, guanine, cytosine, uracil and thymine.

[0055] Non-limitative examples of non-natural nucleotides include 2-Amino-ATP, 8- Aza-ATP, 2'-Fluoro-dATP, 2’-Fluoro-dCTP, 2’-Fluoro-dGTP, 2’-Fluoro-dUTP, 5-Iodo- CTP, 5-Iodo-UTP, N6-Methyl-ATP, 5-Methyl-CTP, 2’-0-Methyl-ATP, 2'-0-Methyl- CTP, 2'-0-Methyl-GTP, 2’-0-Methyl-UTP, Pseudo-UTP, ITP, 2'-0-Methyl-ITP, Puromycin-TP, Xanthosine-TP, 5-Methyl-UTP, 4-Thio-UTP, 2'-Amino-dCTP, 2'- Amino-dUTP, 2’-Azido-dCTP, 2’-Azido-dUTP, 06-Methyl-GTP, 2-Thio-UTP, Ara-CTP, Ara-UTP, 5,6-Dihydro-UTP, 2-Thio-CTP, 6-Aza-CTP, 6-Aza-UTP, Nl-Methyl-GTP, 2'- 0-Methyl-2-Amino-ATP, 2'-O-Methylpseudo-UTP, Nl-Methyl-ATP, 2'-O-Methyl-5- methyl-UTP, 7-Deaza-GTP, 2’-Azido-dATP, 2’-Amino-dATP, Ara-ATP, 8-Azido-ATP, 5-Bromo-CTP, 5-Bromo-UTP, 2’-Fluoro-dTTP, 3 '-O-Methyl- ATP, 3'-O-Methyl-CTP, 3'-O-Methyl-GTP, 3'-O-Methyl-UTP, 7-Deaza-ATP, 5-AA-UTP, 2'-Azido-dGTP, 2'- Amino-dGTP, 5-AA-CTP, 8-Oxo-GTP, Pseudoiso-CTP, N4-Methyl-CTP, N1 - Methylpseudo-UTP, 5,6-Dihydro-5-Methyl-UTP, N6-Methyl-Amino-ATP, 5-Carboxy- CTP, 5-Formyl-CTP, 5-Hydroxymethyl-UTP, 5-Hydroxymethyl-CTP, Thieno-GTP, 5- Hydroxy-CTP, 5-Formyl-UTP, Thieno-UTP, 2-Amino-dATP, 5-Bromo-dCTP, 5- Bromo-dUTP, 7-Deaza-dATP, 7-Deaza-dGTP, diTP, 5-Propynyl-dCTP, 5 -Propynyl- dUTP, 2'-dUTP, 5-Fluoro-dUTP, 5-Iodo-dCTP, 5-Iodo-dUTP, N6-Methyl-dATP, 5- Methyl-dCTP, O6-Methyl-dGTP, N2-Methyl-dGTP, 8-Oxo-dATP, 8-Oxo-dGTP, 2- Thio-dTTP, 2'-dPTP, 5-Hydroxy-dCTP, 4-Thio-dTTP, 2-Thio-dCTP, 6-Aza-dUTP, 6- Thio-dGTP, 8-Chloro-dATP, 5-AA-dCTP, 5-AA-dUTP, N4-Methyl-dCTP, 2'- deoxyzebularine-TP, 5-Hydroxymethyl-dUTP, 5-Hydroxymethyl-dCTP, 5- Propargylamino-dCTP, 5-Propargylamino-dUTP, 5-Carboxy-dCTP, 5-Formyl-dCTP, 5- Indolyl- AA-dUTP, 5-Carboxy-dUTP, 5-Formyl-dUTP, 3'-dATP, 3'-dGTP, 3'-dCTP, 5- Methyl-3'-dUTP, 3'-dUTP, ddATP, ddGTP, ddUTP, ddTTP, ddCTP, 3'-Azido-ddATP, 3’-Azido-ddGTP, 3’-Azido-ddTTP, 3 Amino-ddATP, 3’-Amino-ddCTP, 3’-Amino- ddGTP, 3 '-Amino-ddTTP, 3'-Azido-ddCTP, 3'-Azido-ddUTP, 5-Bromo-ddUTP, ddITP, (l-Thio)-dATP, (l-Thio)-dCTP, (l-Thio)-dGTP, (l-Thio)-dTTP, (l-Thio)-ATP, (1- Thio)-CTP, (l-Thio)-GTP, (l-Thio)-UTP, (l-Thio)-ddATP, (l-Thio)-ddCTP, (1-Thio)- ddGTP, (l-Thio)-ddTTP, (l-Thio)-3'-Azido-ddTTP, (l-Thio)-ddUTP, (1-Borano)- dATP, (l-Borano)-dCTP, (l-Borano)-dGTP, (l-Borano)-dTTP, Ganciclovir- TP, Cidofovir-DP, 3-methyl-6-amino-5-(r-b-D-2'-deoxyribofuranosyl)-pyrimidin-2 -one, 6- amino-9[(r-b-D-2'-deoxyribofuranosyl)-4-hydroxy-5-(hydroxyme thyl)-oxolan-2-yl]- lH-purin-2-one, 6-amino-3-(r-b-D-2'-deoxyribofuranosyl)-5-nitro-l H-pyridin-2-one and 2-amino-8-(r-b-D-2'-deoxyribofuranosyl)-imidazo-[l,2a]-l,3,5 -triazin-[8H]-4-one.

[0056] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine, uracil, thymine and non-natural nucleotides. In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine, uracil and thymine.

[0057] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine and thymine. In one embodiment, Nl is adenine, N2 is guanine, N3 is cytosine and N4 is thymine. In another embodiment, Nl is adenine, N2 is guanine, N3 is thymine and N4 is cytosine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is thymine and N4 is guanine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is guanine and N4 is thymine. In another embodiment, Nl is adenine, N2 is thymine, N3 is cytosine and N4 is guanine. In another embodiment, Nl is adenine, N2 is thymine, N3 is guanine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is adenine, N3 is cytosine and N4 is thymine. In another embodiment, Nl is guanine, N2 is adenine, N3 is thymine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is adenine and N4 is thymine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is thymine and N4 is adenine. In another embodiment, Nl is guanine, N2 is thymine, N3 is adenine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is thymine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is cytosine, N2 is adenine, N3 is guanine and N4 is thymine. In another embodiment, Nl is cytosine, N2 is adenine, N3 is thymine and N4 is guanine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is adenine and N4 is thymine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is thymine and N4 is adenine. In another embodiment, Nl is cytosine, N2 is thymine, N3 is adenine and N4 is guanine. In another embodiment, Nl is cytosine, N2 is thymine, N3 is guanine and N4 is adenine. In another embodiment, Nl is thymine, N2 is adenine, N3 is guanine and N4 is cytosine. In another embodiment, Nl is thymine, N2 is adenine, N3 is cytosine and N4 is guanine. In another embodiment, N 1 is thymine, N2 is guanine, N3 is adenine and N4 is cytosine. In another embodiment, Nl is thymine, N2 is guanine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is thymine, N2 is cytosine, N3 is adenine and N4 is guanine. In another embodiment, Nl is thymine, N2 is cytosine, N3 is guanine and N4 is adenine.

[0058] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine and uracil. In one embodiment, N 1 is adenine, N2 is guanine, N3 is cytosine and N4 is uracil. In another embodiment, Nl is adenine, N2 is guanine, N3 is uracil and N4 is cytosine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is uracil and N4 is guanine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is guanine and N4 is uracil. In another embodiment, N 1 is adenine, N2 is uracil, N3 is cytosine and N4 is guanine. In another embodiment, N 1 is adenine, N2 is uracil, N3 is guanine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is adenine, N3 is cytosine and N4 is uracil. In another embodiment, Nl is guanine, N2 is adenine, N3 is uracil and N4 is cytosine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is adenine and N4 is uracil. In another embodiment, Nl is guanine, N2 is cytosine, N3 is uracil and N4 is adenine. In another embodiment, Nl is guanine, N2 is uracil, N3 is adenine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is uracil, N3 is cytosine and N4 is adenine. In another embodiment, Nl is cytosine, N2 is adenine, N3 is guanine and N4 is uracil. In another embodiment, Nl is cytosine, N2 is adenine, N3 is uracil and N4 is guanine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is adenine and N4 is uracil. In another embodiment, Nl is cytosine, N2 is guanine, N3 is uracil and N4 is adenine. In another embodiment, Nl is cytosine, N2 is uracil, N3 is adenine and N4 is guanine. In another embodiment, Nl is cytosine, N2 is uracil, N3 is guanine and N4 is adenine. In another embodiment, Nl is uracil, N2 is adenine, N3 is guanine and N4 is cytosine. In another embodiment, Nl is uracil, N2 is adenine, N3 is cytosine and N4 is guanine. In another embodiment, Nl is uracil, N2 is guanine, N3 is adenine and N4 is cytosine. In another embodiment, Nl is uracil, N2 is guanine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is uracil, N2 is cytosine, N3 is adenine and N4 is guanine. In another embodiment, Nl is uracil, N2 is cytosine, N3 is guanine and N4 is adenine. [0059] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of non-natural nucleotides.

[0060] In one embodiment, the x components are nucleic acid molecules selected from the group comprising or consisting of double-stranded DNA molecules, single-stranded DNA molecules, double-stranded RNA molecules, single-stranded RNA molecules, and nucleic acid molecules comprising at least one non-natural nucleotide.

[0061] In one embodiment, the x components are x DNA molecules, preferably x double-stranded DNA molecules.

[0062] In one embodiment, the x components are double stranded DNA molecules. In one embodiment, the x components are single stranded DNA molecules.

[0063] In another embodiment, the x components are double stranded RNA molecules or single stranded RNA molecules. In another embodiment, the x components are nucleic acid molecules comprising at least one non-natural nucleotide.

[0064] In one embodiment, the construction of a plurality of x components, each comprising at least one bioblock, comprises the steps of: selectively capturing x data storage nucleic acid molecules from at least one library of data storage nucleic acid molecules, wherein each data storage nucleic acid molecule comprises at least one bioblock surrounded by regions comprising cleavage sites, cleaving each of the x data storage nucleic acid molecules, thereby releasing the at least one bioblock.

[0065] Within the scope of the present invention, the “data storage nucleic acid molecule” is a molecule, typically a plasmid, comprising at least one bioblock e.g., at least one biooctet), or component according to the invention, wherein each bioblock (e.g., biooctet) or component is flanked by regions comprising cleavage sites. In one embodiment, the data storage nucleic acid molecule comprises or consists of nucleotides selected from the group comprising or consisting of natural and non-natural nucleotides. [0066] Within the scope of the present invention, the term “library of data storage nucleic acid molecules” refers to a definite plurality of data storage nucleic acid molecules as defined herein, wherein each data storage nucleic acid molecule of the library comprises distinct bioblocks (e.g., biooctets) or components.

[0067] As used herein, the term “cleavage site” refers to a nucleotide sequence targeted by an enzyme selected from the group comprising or consisting of restriction enzymes (also referred to as restriction endonucleases), endonucleases, exonucleases, deoxyribonuclease, ribonuclease, nickases, transposases and integrases. In a preferred embodiment, the enzyme is a site-directed enzyme, i.e., an enzyme that recognizes a specific nucleic acid sequence.

[0068] In one embodiment, the cleavage sites are targeted by restriction enzymes. In one embodiment, the cleavage sites are restriction sites. As used herein, the term “restriction site” refers to a nucleotide sequence targeted by a specific restriction enzyme. Non- limitative examples of restriction enzymes include EcoRI, BamHI, Hindlll, Kpnl, Notl, PstI, Smal and Xhol. Restriction enzymes and corresponding restriction sites are well known in the art.

[0069] In another embodiment, the cleavage sites are targeted by enzymes selected from the group comprising or consisting of endonucleases, exonucleases, deoxyribonucleases, ribonucleases, nickases, integrases and transposases.

[0070] In one embodiment, the region comprising cleavage sites comprises a first nucleotide sequence that is recognized by the enzyme, typically a restriction enzyme, and a second nucleotide sequence that is digested, or cleaved, by the enzyme. In one embodiment, the first nucleotide sequence and the second nucleotide sequence are distinct. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are separated by at least one nucleotide. In one embodiment, the digestion of the cleavage site separates the first nucleotide sequence from the second nucleotide sequence.

[0071] In one embodiment, the digestion of the cleavage site produces protruding ends or blunt ends, preferably protruding ends. Within the scope of the present invention, these protruding ends are hereby referred to as “fusion sites”. In one embodiment, the protruding end is 3’ protruding or 5’ protruding. In one embodiment, the nucleotide sequences of the 3 ’ protruding end and the 5 ’ protruding end are complementary.

[0072] In one embodiment, the construction of a plurality of x components, each comprising at least one bioblock (e.g., biooctet), comprises the steps of: selectively capturing n data storage nucleic acid molecules from at least two libraries of data storage nucleic acid molecules, wherein each data storage nucleic acid molecule of each library comprises one bioblock (e.g., biooctet) surrounded by regions comprising cleavage sites, and wherein each library comprises all possible bioblocks of m nucleotides (e.g., all possible biooctets of 8 nucleotides), cleaving each of the n data storage nucleic acid molecules, thereby releasing the n bioblocks (e.g., biooctets).

[0073] In one embodiment, the regions comprising cleavage sites comprises from 2 to 25 nucleotides.

[0074] As used herein, the expression “from 2 to 25 nucleotides” comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 nucleotides.

[0075] In one embodiment, the regions comprising cleavage sites comprises from 2 to 20 nucleotides. In one embodiment, the regions comprising cleavage sites comprises from 2 to 15 nucleotides. In one embodiment, the regions comprising cleavage sites comprises from 2 to 10 nucleotides.

[0076] In one embodiment, the cleavage sites are localized both upstream and downstream of the bioblock (e.g. , biooctet) or component.

[0077] As used herein, the term “upstream” refers to a position:

Adjacent in 5’ of the most 5’ end of the sequence of the bioblock (e.g., biooctet) or component, if the data storage nucleic acid molecule is a single stranded nucleic acid molecule, wherein adjacent means either contiguous or separated by a spacer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides); or

- Adjacent in 5’ of the most 5’ end of the sequence of the bioblock (e.g., biooctet) or component on the positive strand, and in 3 ’ of the most 3 ’ end of the sequence of the bioblock (e.g. , biooctet) or component on the negative strand, if the data storage nucleic acid molecule is a double stranded nucleic acid molecule, wherein adjacent means either contiguous or separated by a spacer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0078] As used herein, the term “downstream” refers to a position:

- Adjacent in 3’ of the most 3’ end of the sequence of the bioblock (e.g., biooctet) or component, if the data storage nucleic acid molecule is a single stranded nucleic acid molecule, wherein adjacent means either contiguous or separated by a spacer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides; or

- Adjacent in 3’ of the most 3’ end of the sequence of the bioblock (e.g., biooctet) or component on the positive strand, and in 5 ’ of the most 5 ’ end of the sequence of the bioblock (e.g. , biooctet) or component on the negative strand, if the data storage nucleic acid molecule is a double stranded nucleic acid molecule, wherein adjacent means either contiguous or separated by a spacer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0079] In one embodiment, the data storage nucleic acid molecule comprises a number of upstream regions comprising cleavage sites that is identical to the number of downstream regions comprising cleavage sites. In one embodiment, the data storage nucleic acid molecule comprises at least 1 upstream region comprising a cleavage site and at least 1 downstream region comprising a cleavage site. In one embodiment, the data storage nucleic acid molecule comprises 1 upstream region comprising a cleavage site and 1 downstream region comprising a cleavage site. In one embodiment, the data storage nucleic acid molecule comprises 2 upstream regions comprising cleavage sites and 2 downstream regions comprising cleavage sites. [0080] In one embodiment, the data storage nucleic acid molecule may comprise at least two distinct cleavage sites, wherein distinct cleavage sites have distinct nucleic acid sequence, preferably wherein distinct cleavage sites are digested by distinct enzymes.

[0081 ] In another embodiment, the upstream cleavage site and the downstream cleavage are similar and cleaved by distinct enzymes. In another embodiment, the upstream cleavage site and the downstream cleavage are similar and cleaved by the same enzyme.

[0082] In a preferred embodiment, the upstream cleavage site and the downstream cleavage site are distinct and cleaved by the same enzyme.

[0083] In one embodiment, the data storage nucleic acid molecule further comprises 2 additional cleavage sites, wherein the first one is localized upstream of the bioblocks (e.g. , biooctets) or components and the second one is localized downstream of the bioblocks (e.g., biooctets) or components.

[0084] In one embodiment, the 2 additional cleavage sites are distinct and cleaved by the same enzyme. In another embodiment, the 2 additional cleavage sites are distinct and cleaved by distinct enzymes. In another embodiment, the 2 additional cleavage sites are similar and cleaved by the same enzyme. In another embodiment, the 2 additional cleavage sites are similar and cleaved by distinct enzymes.

[0085] In one embodiment, the 2 additional cleavage sites are distinct from the other cleavage sites comprised on the data storage nucleic acid molecule and are cleaved by enzymes distinct from those cleaving the cleavage sites comprised on the data storage nucleic acid molecule. In another embodiments, the 2 additional cleavage sites are similar from the other cleavage sites comprised on the data storage nucleic acid molecule and are cleaved by enzymes similar to those cleaving the cleavage sites comprised on the data storage nucleic acid molecule.

[0086] In one embodiment, the bioblocks (e.g., biooctet) or components are considered released when at least one upstream cleavage site and at least one downstream cleavage site are cleaved (i.e., digested or cut). [0087] In one embodiment, a released bioblock (e.g., biooctet) comprises (i) one bioblock (e.g., biooctet), (ii) part of the closest upstream cleavage site, i.e., the upstream fusion site, and (iii) part of the closest downstream cleavage site, i.e., the downstream fusion site. In one embodiment, the part of the closest upstream cleavage site, i.e., the upstream fusion site, is a protruding end (e.g., 3’ protruding end). In one embodiment, the part of the closest downstream cleavage site, i.e., the downstream fusion site, is a protruding end (e.g., 5’ protruding end).

[0088] In one embodiment, a released component comprises (i) at least one bioblock (e.g., biooctet), (ii) part of the closest upstream cleavage site, i.e., the upstream fusion site, and (iii) part of the closest downstream cleavage site, i.e., the downstream fusion site. In a preferred embodiment, a released component comprises (i) y bioblocks e.g., biooctets), (ii) part of the closest upstream cleavage site, i. e. , the upstream fusion site, and (iii) part of the closest downstream cleavage site, i.e., the downstream fusion site.

[0089] In one embodiment, assembling together a plurality of x components involves releasing bioblocks e.g., biooctets) or components. In one embodiment, releasing bioblocks e.g., biooctets) or components involves using either one enzyme or two distinct enzymes.

[0090] In one embodiment, each of the region surrounding each bioblock e.g. , biooctet) comprises a site for a restriction enzyme, and step (d) of the method of the invention comprises a step of digesting each of the x data storage nucleic acid molecules with one or two restriction enzymes.

[0091] In another embodiment, each of the region surrounding each bioblock e.g., biooctet) comprises a site for a restriction enzyme, and step (d) of the method of the invention comprises a step of digesting each of the x data storage nucleic acid molecules with two restriction enzymes.

[0092] In one embodiment, digestion of the upstream restriction site produces a 3’ protruding end or a 5’ protruding end, digestion of the downstream restriction site produces a 3 ’ protruding end or a 5 ’ protruding end. In one embodiment, the nucleotide sequences of the 3 ’ protruding end and the 5 ’ protruding end are complementary. [0093] In one embodiment, the restriction site comprises a first nucleotide sequence that is recognized by the restriction enzyme, and a second nucleotide sequence that is digested, or cleaved, by the enzyme. In one embodiment, the first nucleotide sequence and the second nucleotide sequence are distinct. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are separated by at least one nucleotide. In one embodiment, the digestion of the restriction site separates the first nucleotide sequence from the second nucleotide sequence.

[0094] In one embodiment, the restriction enzymes are selected from the group comprising or consisting of type I, type II, type III, type IV or type V restriction enzymes, or combinations thereof. In one embodiment, the restriction enzyme is a type II restriction enzyme. In one embodiment, the type II restriction enzymes are selected from the group comprising or consisting of type II S, type II G, type II B, type II T and/or type II C restriction enzymes, or combination thereof, preferably type II S and/or type II G, more preferably type II S. Non- limitative examples of type II S restriction enzymes include Bsal, BbsI, BsmBI, FokI, Alw26I, Bbvl, BsrI, Earl, HphI, MboII, SfaNI and Tthl 1 II. In one embodiment, the restriction enzymes are Bsal and/or BbsI and/or BsmBI.

[0095] In one embodiment, the restriction enzymes are modified. In one embodiment, the restriction enzymes comprise at least one mutation in their amino acid sequence compared to the unmodified (or wild type) amino acid sequence. In one embodiment, the restriction enzymes are post-translationally modified.

[0096] In certain embodiments, the enzyme recognition sites consist of a nucleotide sequence selected from GGTCTC and CGTCTC.

[0097] In one embodiment, the cleavage sites comprise a nucleotide sequence selected from the group comprising or consisting of GTAG, TGAC, TCAG, AATA, TCAA, CTTC, AGTA, ACTG, CACA, CCAG, CAAA, GACC, ACTC, CCAC, GAAC, GCAC, CGGC, CGTA, GTAA, CAAC, GCTA, CCGA, ACGA, AGAA, TAAA, AGCG, ACCT, AACA, GGCA, ACGC, AATC, CGAG, TCCA, CCTA, CTAA, GGGA, AAGG, AAAC, CTAC, and GAGA. In one embodiment, these sequences are protruding ends. [0098] In one embodiment, the fusion sites comprise a nucleotide sequence selected from the group comprising or consisting of GTAG, TGAC, TCAG, AATA, TCAA, CTTC, AGTA, ACTG, CACA, CCAG, CAAA, GACC, ACTC, CCAC, GAAC, GCAC, CGGC, CGTA, GTAA, CAAC, GCTA, CCGA, ACGA, AGAA, TAAA, AGCG, ACCT, AACA, GGCA, ACGC, AATC, CGAG, TCCA, CCTA, CTAA, GGGA, AAGG, AAAC, CTAC, and GAGA.

[0099] In one embodiment, the cleavage sites comprise a nucleotide sequence selected from the group comprising or consisting of GTAG, TGAC, TCAG. In one embodiment, the cleavage sites comprise a nucleotide sequence selected from the group comprising or consisting of AATA, TCAA, CTTC, AGTA, ACTG, CACA, CCAG, CAAA, GACC, ACTC, CCAC, GAAC, GCAC, CGGC, CGTA, GTAA, CAAC, GCTA, CCGA, ACGA, AGAA, TAAA, AGCG, ACCT, AACA, GGCA, ACGC, AATC, CGAG, TCCA, CCTA, CTAA and GGGA. In one embodiment, the cleavage sites comprise a nucleotide sequence selected from the group comprising or consisting of AATA, AAGG, AAAC, TAAA, ACGA, ACTG, AGCG, GCTA, GGCA, ACCT, CGTA, AACA, CTAC, GAGA, CCAG, AGAA and GCAC.

[0100] In one embodiment, the fusion sites comprise a nucleotide sequence selected from the group comprising or consisting of GTAG, TGAC, TCAG. In one embodiment, the fusion sites comprise a nucleotide sequence selected from the group comprising or consisting of AATA, TCAA, CTTC, AGTA, ACTG, CACA, CCAG, CAAA, GACC, ACTC, CCAC, GAAC, GCAC, CGGC, CGTA, GTAA, CAAC, GCTA, CCGA, ACGA, AGAA, TAAA, AGCG, ACCT, AACA, GGCA, ACGC, AATC, CGAG, TCCA, CCTA, CTAA and GGGA. In one embodiment, the fusion sites comprise a nucleotide sequence selected from the group comprising or consisting of AATA, AAGG, AAAC, TAAA, ACGA, ACTG, AGCG, GCTA, GGCA, ACCT, CGTA, AACA, CTAC, GAGA, CCAG, AGAA and GCAC.

[0101] In one embodiment, step (e) comprises one or several assembling steps using overlap-extension polymerase chain reaction (PCR), polymerase cycling assembly, sticky end ligation, biobricks assembly, golden gate assembly, Gibson assembly, recombinase assembly, ligase cycling reaction, template directed ligation, in vivo assembly or any other DNA assembly protocol.

[0102] In one embodiment, step (e) comprises one or several assembling steps using overlap PCR. In one embodiment, step (e) comprises one or several assembling steps using polymerase cycling assembly. In one embodiment, step (e) comprises one or several assembling steps using sticky end ligation. In one embodiment, step (e) comprises one or several assembling steps using biobricks assembly. In one embodiment, step (e) comprises one or several assembling steps using golden gate assembly. In one embodiment, step (e) comprises one or several assembling steps using Gibson assembly. In one embodiment, step (e) comprises one or several assembling steps using recombinase assembly. In one embodiment, step (e) comprises one or several assembling steps using ligase cycling reaction. In one embodiment, step (e) comprises one or several assembling steps using template directed ligation. In one embodiment, step (e) comprises one or several assembling steps using in vivo assembly.

[0103] In one embodiment, step (e) comprises using a ligase.

[0104] In a preferred embodiment, the cleavage of the regions comprising cleavage sites produces protruding ends, also referred to as fusion sites. In a preferred embodiment, the closest fusion site on one end (e.g., 3’ end) of the first bioblock (e.g., biooctet) or component, and the closest fusion site on the other end (e.g., 5’ end) of the second bioblock (e.g. , biooctet) or component are complementary.

[0105] In one embodiment, the assembly of components comprising at least one bioblock (e.g., biooctets) necessitates or is facilitated by the complementarity between:

- the closest fusion site on one end (e.g., 3’ end) of a first bioblock (e.g., biooctet), and

- the closest fusion site on the other end (e.g., 5’ end) of a second bioblock (e.g., biooctet).

[0106] In one embodiment, the nucleotide sequence recognized by the enzyme is not comprised on the nucleotide sequence digested by the enzyme. In one embodiment, upon digestion of the cleavage site, the nucleotide sequence recognized by the enzyme is lost, i.e., it is separated from the cleaved sequence. In one embodiment, the cleavage sites between 2 bioblocks (e.g., between 2 biooctets) or 2 components do not comprise the nucleotide sequence recognized by the enzyme.

[0107] In one embodiment, an assembled component comprising y bioblocks (e.g., biooctets) comprises or consists of:

- y bioblocks (e.g. , biooctets) in a fixed order, y+1 fusion sites flanking the bioblocks.

[0108] In one embodiment, an assembled component comprising y bioblocks (e.g., biooctets) comprises or consists of:

- y bioblocks (e.g. , biooctets) in a fixed order, y+1 fusion sites flanking the bioblocks, and

2 regions comprising cleavage sites, wherein the regions comprising the cleavage sites are localized at the furthest 5 ’ end and the furthest 3 ’ end of the component.

[0109] The present invention further relates to a data storage nucleic acid molecule comprising at least one bioblock, a bioblock consisting of a nucleic acid sequence consisting of m nucleotides assigned to positions 0 to m-1, wherein a bioblock is formed of at least 2 and at most 4 (i.e., 2, 3 or 4) distinct nucleotides

- nucleotides at even positions may be selected from a first and a second nucleotide, and nucleotides at odd positions may be selected from a third and a fourth nucleotide, said first, second, third and fourth nucleotides being distinct.

[0110] In one embodiment, the first, second, third and fourth nucleotides are referred to as Nl, N2, N3 and N4, respectively.

[0111] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine, uracil, thymine and non-natural nucleotides, wherein N 1 , N2, N3 and N4 are distinct nucleotides. In one embodiment, N 1 , N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine, uracil and thymine, wherein Nl, N2, N3 and N4 are distinct nucleotides.

[0112] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine and thymine, wherein Nl, N2, N3 and N4 are distinct nucleotides. In one embodiment, Nl is adenine, N2 is guanine, N3 is cytosine and

N4 is thymine. In another embodiment, Nl is adenine, N2 is guanine, N3 is thymine and N4 is cytosine. In another embodiment, N 1 is adenine, N2 is cytosine, N3 is thymine and N4 is guanine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is guanine and N4 is thymine. In another embodiment, Nl is adenine, N2 is thymine, N3 is cytosine and N4 is guanine. In another embodiment, Nl is adenine, N2 is thymine, N3 is guanine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is adenine, N3 is cytosine and N4 is thymine. In another embodiment, Nl is guanine, N2 is adenine, N3 is thymine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is adenine and N4 is thymine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is thymine and N4 is adenine. In another embodiment, Nl is guanine, N2 is thymine, N3 is adenine and N4 is cytosine. In another embodiment, N 1 is guanine, N2 is thymine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is cytosine, N2 is adenine, N3 is guanine and N4 is thymine. In another embodiment, Nl is cytosine, N2 is adenine, N3 is thymine and N4 is guanine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is adenine and N4 is thymine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is thymine and N4 is adenine. In another embodiment, Nl is cytosine, N2 is thymine, N3 is adenine and N4 is guanine. In another embodiment, Nl is cytosine, N2 is thymine, N3 is guanine and N4 is adenine. In another embodiment, Nl is thymine, N2 is adenine, N3 is guanine and N4 is cytosine. In another embodiment, N 1 is thymine, N2 is adenine, N3 is cytosine and N4 is guanine. In another embodiment, Nl is thymine, N2 is guanine, N3 is adenine and N4 is cytosine. In another embodiment, N 1 is thymine, N2 is guanine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is thymine, N2 is cytosine, N3 is adenine and N4 is guanine. In another embodiment, Nl is thymine, N2 is cytosine, N3 is guanine and N4 is adenine. [0113] In one embodiment, Nl, N2, N3 and N4 are selected from the group comprising or consisting of adenine, guanine, cytosine and uracil, wherein Nl, N2, N3 and N4 are distinct nucleotides. In one embodiment, Nl is adenine, N2 is guanine, N3 is cytosine and N4 is uracil. In another embodiment, N 1 is adenine, N2 is guanine, N3 is uracil and N4 is cytosine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is uracil and N4 is guanine. In another embodiment, Nl is adenine, N2 is cytosine, N3 is guanine and N4 is uracil. In another embodiment, Nl is adenine, N2 is uracil, N3 is cytosine and N4 is guanine. In another embodiment, Nl is adenine, N2 is uracil, N3 is guanine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is adenine, N3 is cytosine and N4 is uracil. In another embodiment, Nl is guanine, N2 is adenine, N3 is uracil and N4 is cytosine. In another embodiment, Nl is guanine, N2 is cytosine, N3 is adenine and N4 is uracil. In another embodiment, Nl is guanine, N2 is cytosine, N3 is uracil and N4 is adenine. In another embodiment, Nl is guanine, N2 is uracil, N3 is adenine and N4 is cytosine. In another embodiment, Nl is guanine, N2 is uracil, N3 is cytosine and N4 is adenine. In another embodiment, N 1 is cytosine, N2 is adenine, N3 is guanine and N4 is uracil. In another embodiment, Nl is cytosine, N2 is adenine, N3 is uracil and N4 is guanine. In another embodiment, Nl is cytosine, N2 is guanine, N3 is adenine and N4 is uracil. In another embodiment, Nl is cytosine, N2 is guanine, N3 is uracil and N4 is adenine. In another embodiment, Nl is cytosine, N2 is uracil, N3 is adenine and N4 is guanine. In another embodiment, N 1 is cytosine, N2 is uracil, N3 is guanine and N4 is adenine. In another embodiment, Nl is uracil, N2 is adenine, N3 is guanine and N4 is cytosine. In another embodiment, Nl is uracil, N2 is adenine, N3 is cytosine and N4 is guanine. In another embodiment, Nl is uracil, N2 is guanine, N3 is adenine and N4 is cytosine. In another embodiment, Nl is uracil, N2 is guanine, N3 is cytosine and N4 is adenine. In another embodiment, Nl is uracil, N2 is cytosine, N3 is adenine and N4 is guanine. In another embodiment, Nl is uracil, N2 is cytosine, N3 is guanine and N4 is adenine.

[0114] In some embodiments, Nl, N2, N3 and N4 are non-natural nucleotides as described hereinabove, wherein Nl, N2, N3 and N4 are distinct nucleotides. [0115] In one embodiment, the data storage nucleic acid molecule is a double-stranded molecule, preferably a DNA molecule.

[0116] In one embodiment, the double stranded nucleic acid molecule is circular or linear, preferably circular. In one embodiment, the data storage nucleic acid molecule is a linear sequence that has been circularized. Method to circularize a DNA sequence are known in the art.

[0117] In one embodiment, the data storage nucleic acid molecule is a plasmid, a cosmid, a fosmid, a prokaryotic chromosome (e.g., bacterial artificial chromosome) or a eukaryotic chromosome (e.g., yeast artificial chromosome or human artificial chromosome).

[0118] In a preferred embodiment, the data storage nucleic acid molecule is a plasmid. In another embodiment, the data storage nucleic acid molecule is a cosmid. In another embodiment, the data storage nucleic acid molecule is a fosmid. In another embodiment, the data storage nucleic acid molecule is a prokaryotic chromosome. In another embodiment, the data storage nucleic acid molecule is a eukaryotic chromosome.

[0119] In one embodiment, in the data storage nucleic acid molecule, each of the bioblocks (e.g., biooctets) or component is surrounded by regions comprising at least one cleavage site. In one embodiment, in the data storage nucleic acid molecule, each of the bioblocks (e.g., biooctets) or component is surrounded by regions comprising one cleavage site. In another embodiment, in the data storage nucleic acid molecule, each of the bioblocks (e.g., biooctets) or component is surrounded by regions comprising two cleavage sites, wherein the cleavage sites within the same region are distinct.

[0120] In one embodiment, the digestion of the regions comprising cleavage site by the restriction enzymes produces protruding end or blunt ends, preferably protruding ends (i.e., fusion sites). In one embodiment, protruding ends are 3’ protruding or 5’ protruding.

[0121] In one embodiment, a data storage nucleic acid molecule comprises at least one component, and each of the component is surrounded by regions comprising one or more cleavage sites. [0122] In one embodiment, the data storage nucleic acid molecule is replicative.

[0123] As used herein, the “replicative” property of the data storage nucleic acid molecule according to the invention refers to its ability to be duplicated one or more time(s) in vivo in a living organism, in particular by a polymerase, more particularly by a DNA polymerase.

[0124] In one embodiment, the assessment of the replicative property of a nucleic acid molecule may be performed according to any standard method from the state of the art, or a method derived therefrom. Illustratively, the replicative property may be assessed by the increase of the number of copies of said nucleic acid molecules in/by a living organism and/or the ability of the living organism to transfer the nucleic acid to its progeny.

[0125] In one embodiment, the living organism is a microorganism, in particular a bacterium, a microalga, an archaeon, a fungus, a phage, a virus or a yeast. In one embodiment, the living organism is a prokaryote. Non-limitative examples of prokaryotes according to the invention include bacteria, such as actinobacteria, chlamydiales, cyanobacteria, firmicutes, proteobacteria, spirochetes, thermotogales; and archaea, such as euarchaeota, crenarchaeota. In one embodiment, the living organism is a bacterium, preferably Escherichia coli, more preferably Escherichia coli strain DH5a.

[0126] In certain embodiments, the living organism is a eukaryote. Non-limitative examples of eukaryotes according to the invention include protozoa, algae, plants, fungi, animals and their respective cells thereof.

[0127] In order to be replicated, the data storage nucleic acid molecule according to the invention possesses at least one origin of replication, namely one or more sequence(s) of nucleotides recognized by a replication initiation machinery. Illustratively, archaeon and bacterial origins of replication include oriC. In practice, most bacteria may have a unique origin of replication; an archaeon may have one or more origin(s) of replication; a eukaryote may have multiple origins of replication, in particular in the form of centromeres. Within the scope of the instant invention, the term “multiple origins of replication” refers to at least 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200 origins of replication per nucleic acid molecule. [0128] In one embodiment, the data storage nucleic acid molecule comprises or consists of (i) at least one component as described hereinabove, and (ii) at least one origin of replication.

[0129] In one embodiment, the data storage nucleic acid molecule does not comprise a promoter region. In one embodiment, the data storage nucleic acid molecule does not comprise a biological coding sequence.

[0130] In one embodiment, the data storage nucleic acid molecule is non-coding.

[0131] In one embodiment, the size of the data storage nucleic acid molecule is comprised between 100 base pairs (bp) and 1.10 ⁶ bp. As used herein, the expression “between 100 base pairs (bp) and 10 ⁶ bp” comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10 ⁴ , 10 ⁵ , and 10 ⁶ bp.

[0132] In some embodiments, the data storage nucleic acid molecule further comprises one or more regions carrying metadata, i.e., information that do not encode digital information. Typically, these regions are termed “metadata bioblocks” (e.g., metadata biooctet).

[0133] In some embodiments, the metadata region comprises or consists of at least one barcoding region. As used herein, the term “barcoding region” refers to a bioblock (e.g., biooctet) added at the beginning of a component, or group of components. Typically, the barcode encodes a number (e.g., 0, 1, 2, 3, 4 and the like) using the same encoding system as the bioblocks, and the numbering system allows to label the components, or group of components, in a definite order.

[0134] In some embodiments, the metadata region comprises or consists of a “end of file” signal. As used herein, the term “end of file signal” refers to a special bioblock (e.g., biooctet) with a predefined sequence that is not shared with any other bioblock, that is localized at the end of the sequence. Typically, the “end of file” signal indicates the end of the region encoding digital data of the file.

[0135] In some embodiments, the metadata region comprises or consists of at least one barcoding region and one “end of file signal”, as described hereinabove. [0136] The present invention further relates to a library comprising a plurality of data storage nucleic acid molecules according to the invention, wherein each of the data storage nucleic acid molecule of the library contains one bioblock (e.g. , biooctet), wherein each data storage nucleic acid molecule of the library comprises the same surrounding regions comprising cleavage sites and wherein the library contains all possible bioblocks of m nucleotides.

[0137] In one embodiment, each data storage molecule of the library comprises exactly one bioblock (e.g., biooctet). In one embodiment, the total number of data storage nucleic acid molecules in the library is equal to 2 ^m . In one embodiment, m=8; thus, the size of the library is 256 data storage nucleic acid molecules.

[0138] In one embodiment, each data storage molecule of the library comprises a distinct bioblock (e.g., biooctet). In practice, a library comprises 2 ^m distinct bioblocks (e.g., biooctets).

[0139] In one embodiment, two distinct libraries comprise distinct bioblocks (e.g., biooctets). In another embodiment, two distinct libraries may comprise at least one common (i.e., identical) bioblock (e.g., biooctet). In certain embodiments, two distinct libraries comprise more than 2 ^m distinct bioblocks (e.g., biooctets).

[0140] In another embodiment, each data storage molecule comprises components according to the invention, wherein each component comprises more than one bioblock (e.g., biooctet). In one embodiment, each data storage molecule of the library comprises at least 1 component. In one embodiment, each data storage molecule of the library comprises a distinct component. In one embodiment, two distinct libraries comprise distinct components. In another embodiment, two distinct libraries may comprise at least one common (i.e., identical) component.

[0141] In one embodiment, each data storage molecule of the library comprises from 1 to 32 components. As used herein, the expression from 1 to 32 encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. In one embodiment, each data storage molecule of the library comprises from 2 to 32 components. In one embodiment, each data storage molecule of the library comprises from 4 to 32 components. In one embodiment, each data storage molecule of the library comprises from 8 to 32 components. In one embodiment, each data storage molecule of the library comprises from 16 to 32 components. In one embodiment, each data storage molecule of the library comprises from 1 to 16 components. In one embodiment, each data storage molecule of the library comprises from 1 to 8 components. In one embodiment, each data storage molecule of the library comprises from 1 to 4 components. In one embodiment, each data storage molecule of the library comprises from 1 to 2 components.

[0142] In another embodiment, each data storage molecule of the library comprises more than 32 components.

[0143] In some embodiments, libraries comprising data storage molecule comprising at least one component are assembled using the bioblocks (e.g., biooctets) released from at least one library comprising data storage molecule comprising exactly one bioblock (e.g., biooctet), using the method as disclosed in the present invention. In practice, a nucleic acid molecule comprising exactly one couple of cleavage sites identical to the cleavage sites flanking the bioblocks (e.g. , biooctets), herein referred to as acceptor molecule, is digested using at least one enzyme, preferably one enzyme, and is assembled with at least one bioblock (e.g., biooctet) using the method as described hereinabove.

[0144] In some embodiments, libraries comprising data storage molecules comprising more than one component are assembled using the components released from at least one library comprising data storage molecule comprising exactly one component, using the method as disclosed in the present invention.

[0145] In one embodiment, the regions comprising cleavage sites comprised on each data storage molecule of the library are identical.

[0146] In one embodiment, data storage molecules of distinct libraries comprise distinct regions comprising cleavage sites.

[0147] In one embodiment, data storage nucleic acid molecules of distinct libraries comprise identical regions comprising cleavage sites, wherein the bioblocks (e.g., biooctets) or components comprised in the data storage molecule of the first library are not used to assemble components comprised in the data storage molecule of the second library, and wherein the bioblocks (e.g., biooctets) or components comprised in the data storage molecule of the second library are not used to assemble components comprised in the data storage molecule of the first library.

[0148] In one embodiment, components may be assembled using bioblocks (e.g., biooctets) or components from more than one library.

[0149] In one embodiment, the data storage nucleic acid molecules comprised in the library are identified and labelled according to:

- the nucleic acid sequence of the bioblocks (e.g., biooctets) and/or components they comprise, and/or

- the nucleic acid sequence, or region, comprising cleavage sites surrounding the bioblocks (e.g., biooctets) and/or components, and/or

- the encoding system used to convert digital subsequences comprising m bits (i. e. , value and position of the bits) into bioblocks, according to the method of the invention. In one embodiment, the encoding system is displayed in the form “(N 1 , N2, N3, N4)”, “(Nl, N2, N3)” or “(Nl, N2)”.

[0150] In one embodiment, the labelling information is digital and/or physical. In one embodiment, the labelling information is stored in at least one database.

[0151] In one embodiment, data storage nucleic acid molecules comprised in the library are labelled using a code or an identifier that does not provide any information regarding the content of the data storage nucleic acid molecules. In one embodiment the information regarding the sequence of the data storage nucleic acid molecules and the encoding system are retrieved by searching for the corresponding code or identifier within the at least one database.

[0152] In a preferred embodiment, the data storage nucleic acid molecules comprised in the library are stored separately. [0153] In one embodiment, the data storage nucleic acid molecules of a library are stored at a temperature suitable for preventing nucleic acid degradation. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised from 4 °C to -200 °C. As used herein, the expression “from 4 °C to -200 °C” encompasses 4, 3, 2, 1, 0, -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -30, -40, -50, -60, -70, -80, -90, -100, -120, -140, -160, -180, -200 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between 4 °C and -80 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between 4 °C and -20 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between 4 °C and 0 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between 0 °C and -200 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between -20 °C and -200 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature comprised between -80 °C and -200 °C. In one embodiment, the data storage nucleic acid molecules comprised in the library are stored at a temperature of -196 °C

[0154] In one embodiment, the data storage nucleic acid molecules comprised in the library are stored in a suitable solvent. Suitable solvents for nucleic acid storage are known in the art. Non limitative examples of solvents used for nucleic acid storage include aqueous solvents such as demineralized water or biological buffers (e.g., phosphate-buffered saline, Tris-HCl).

[0155] In one embodiment, the data storage nucleic acid molecules comprised in the library are lyophilized.

[0156] The present invention further relates to a nucleic acid-based data storage system comprising at least two libraries according to the invention.

[0157] In one embodiment, the data storage nucleic acid molecules of the at least two libraries comprise bioblocks (e.g. , biooctets) and/or components. In one embodiment, the data storage nucleic acid molecules of the at least two libraries comprise bioblocks (e.g., biooctets).

[0158] In one embodiment, the nucleic acid-based data storage system is for storing data comprised in a digital sequence as described hereinabove. In one embodiment, the conversion of information carried by the digital sequence into the nucleic acid-based data storage system, i.e., encoding, is performed using the method of the present disclosure.

[0159] In one embodiment, the digital data consist of binary digital data. In practice, converting digital data into a nucleic acid molecule may be performed automatically by a suitable software in silico.

[0160] In one embodiment, the data comprised in a digital sequence is stored on at least one data storage nucleic acid molecule, wherein the at least one data storage nucleic acid molecule is assembled using the method according to the invention, from libraries according to the invention.

[0161] In one embodiment, nucleic acid-based data storage system can store the equivalent of an amount of information comprised from 2 to 10 ²¹ bytes. As used herein, the expression “from 2 to 10 ²¹ bytes” comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 128, 256, 500, 512, 1000, 1024, 2048, 4096, 8192, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , 10 ¹⁶ , 10 ¹⁷ , 10 ¹⁸ , 10 ¹⁹ , 10 ² °, 10 ²¹ bytes.

[0162] Another object of the present invention is a computer software for implementing the use and method for storing digital data.

[0163] In one embodiment, the method of the invention is implemented with a microprocessor comprising a software configured to assign to digital data at least one nucleic acid molecule according to the invention. In some embodiments, the software is configured to prevent that the sequence of the composite nucleic acid molecule according to the invention would encode one or more RNA(s), preferably would not encode any mRNA(s). In some embodiments, the software is configured to prevent that the sequence of the composite nucleic acid molecule according to the invention would comprise one or more initiation codon(s) in all 6 reading frames. In some embodiments, the software is configured to prevent that the sequence of the composite nucleic acid molecule according to the invention would comprise one or more specific restriction site(s). In some embodiments, the software is configured to prevent that the sequence of the composite nucleic acid molecule according to the invention would comprise one or more repeat(s) of at least 5 identical nucleotides.

[0164] In one embodiment, information can be retrieved from the nucleic acid-based data storage system by sequencing the at least one nucleic acid molecule. Methods of sequencing nucleic acid molecules, in particular high throughput sequencing, are known in the art and comprise, inter alia, Illumina (sequencing by synthesis), single-molecule real-time (SMRT) sequencing, nanopore sequencing (e.g., sequencing solutions from Oxford Nanopore Technologies), sequencing by ligation or sequencing by chain termination (Sanger method).

[0165] In one embodiment, converting the data retrieved from the data storage system into digital data further requires to obtain:

- the encoding system used to convert digital subsequences comprising m bits (z. e. , value and position of the bits) into bioblocks, according to the method of the invention,

- the sequence of the regions comprising cleavage sites, the position and type of metadata bioblocks, the value of m, the value of n and x.

[0166] In one embodiment, converting the data retrieved from the data storage system into digital data results in the retrieval of a sequence of bytes comprising m bits. In one embodiment, converting the data retrieved from the data storage system into digital data results in the retrieval of a sequence of octets. [0167] In one embodiment, the information required to convert the data retrieved from the data storage system into digital data is stored in at least one database. In another embodiment, the information required to convert the data retrieved from the data storage system into digital data is stored in metadata bioblocks.

[0168] In one embodiment, the conversion of data contained in the data storage system into digital data is automated, i.e., by a suitable software or program. In practice, a program in which are entered (i) the sequence of the at least one data storage nucleic acid molecule and (ii) the information required to convert the data retrieved from the data storage system into digital data (i.e., the encoding system, the sequence of the cleavage sites, the position and type of metadata bioblocks, the value of m and the value of both n and x), provides a sequence of bytes comprising m bits, optionally a sequence of octets. Typically, the nucleotides corresponding to the cleavage sites are skipped by the program.

[0169] In one embodiment, said sequence of bytes, optionally octets, is read as such. In one embodiment, said sequence of bytes, optionally octets, is first converted to a file format as described in the present disclosure. In one embodiment, the converted file is read by an adequate program.

[0170] Another object of the present invention is a computer software for implementing the use and method for retrieving digital data. In one embodiment, the method of the invention is implemented with a microprocessor comprising a software configured to convert at least one nucleic acid sequence into digital data, using the method as described hereinabove.

BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1 is a schematic representing the pipeline of the complete method for encoding digital data.

[0172] Figure 2 is a schematic representing the design of library A and library B, with each plasmid comprising one bioblock.

[0173] Figure 3 is a schematic representing the design of BioblockX2 plasmids. [0174] Figure 4 is a schematic representing the design of BioblockX64 plasmids.

[0175] Figure 5 is a schematic representing the design of Biob lockX 1024 plasmids.

EXAMPLES [0176] The present invention is further illustrated by the following example of 8-bit biodata encoding

Materials and Methods

[0177] Practical biodata encoding of a text file containing the poem Liberte written by Paul Eluard in 1942 (Table 1).

Liberte

Sur mes cahiers d’ecolier

Sur mon pupitre et les arbres

Sur le sable sur la neige

J’ecris ton nom

Sur toutes les pages lues

Sur toutes les pages blanches

Pierre sang papier ou cendre

J’ecris ton nom

Sur les images dorees

Sur les armes des guerriers

Sur la couronne des rois

J’ecris ton nom

Sur la jungle et le desert

Sur les nids sur les genets

Sur 1’echo de mon enfance

J’ecris ton nom

Sur les merveilles des nuits

Sur le pain blanc des joumees

Sur les saisons fiancees

J’ecris ton nom Sur tous mes chiffons d’azur

Sur 1’ etang soleil moisi

Sur le lac lune vivante J’ecris ton nom

Sur les champs sur 1’horizon Sur les ailes des oiseaux Et sur le moulin des ombres J’ecris ton nom

Sur chaque bouffee d’aurore Sur la mer sur les bateaux Sur la montagne demente J’ecris ton nom

Sur la mousse des nuages Sur les sueurs de I’orage Sur la pluie epaisse et fade J’ecris ton nom

Sur les formes scintillantes

Sur les cloches des couleurs

Sur la verite physique J’ecris ton nom

Sur les sentiers eveilles

Sur les routes deployees

Sur les places qui debordent

J’ecris ton nom

Sur la lampe qui s’allume Sur la lampe qui s’eteint Sur mes maisons reunies J’ecris ton nom

Sur le fruit coupe en deux Du miroir et de ma chambre Sur mon lit coquille vide J’ecris ton nom

Sur mon chien gourmand et tendre Sur ses oreilles dressees Sur sa pate maladroite J’ecris ton nom

Sur le tremplin de ma porte Sur les objets familiers Sur le flot du feu beni J’ecris ton nom

Sur toute chair accordee

Sur le front de mes amis

Sur chaque main qui se tend

J’ecris ton nom

Sur la vitre des surprises Sur les levres attentives Bien au-dessus du silence J’ecris ton nom

Sur mes refuges detruits Sur mes phares ecroules Sur les murs de mon ennui J’ecris ton nom

Sur 1’ absence sans desir

Sur la solitude nue

Sur les marches de la mort J’ecris ton nom

Sur la sante revenue

Sur le risque disparu

Sur 1’espoir sans souvenir J’ecris ton nom

Et par le pouvoir d’un mot Je recommence ma vie Je suis ne pour te connaitre Pour te nommer

Liberte.

Paul Eluard Encode par le Centre National de la Recherche Scientifique et Sorbonne Universite a Paris, France, 2021.

Avec la permission des Editions de Minuit.

Table 1: Original text, poem Liberte by Paul Eluard

[0178] The text is encoded using the ISO8859-1 standard, also known as Latin- 1, to generate file A comprising 2358 octets (Table 2). File A is compressed as a 7z archive with the LZMA2 algorithm to generate file B comprising 1137 octets (Table 3). File B corresponds to a digital sequence formed of a plurality of 9096 bits. This digital sequence is subdivided into n=l 137 digital subsequences each comprising m=8 bits. Each of these 1137 digital subsequences of 8 bits are converted into a bioblock of m=8 nucleotides named a biooctet.

010011000110100101100010011001010111001001110100111010010 0001101000010100000110100 001010000011010000101001010011011101010111001000100000011011 0101100101011100110010 000001100011011000010110100001101001011001010111001001110011 0010000001100100001001 111110100101100011011011110110110001101001011001010111001000 0011010000101001010011 011101010111001000100000011011010110111101101110001000000111 0000011101010111000001 101001011101000111001001100101001000000110010101110100001000 0001101100011001010111 001100100000011000010111001001100010011100100110010101110011 0000110100001010010100 110111010101110010001000000110110001100101001000000111001101 1000010110001001101100 011001010010000001110011011101010111001000100000011011000110 0001001000000110111001 100101011010010110011101100101000011010000101001001010001001 1111101001011000110111 001001101001011100110010000001110100011011110110111000100000 0110111001101111011011 010000110100001010000011010000101000001101000010100101001101 1101010111001000100000 011101000110111101110101011101000110010101110011001000000110 1100011001010111001100 100000011100000110000101100111011001010111001100100000011011 0001110101011001010111 001100001101000010100101001101110101011100100010000001110100 0110111101110101011101 000110010101110011001000000110110001100101011100110010000001 1100000110000101100111 011001010111001100100000011000100110110001100001011011100110 0011011010000110010101 110011000011010000101001010000011010010110010101110010011100 1001100101001000000111 001101100001011011100110011100100000011100000110000101110000 0110100101100101011100 100010000001101111011101010010000001100011011001010110111001 1001000111001001100101 000011010000101001001010001001111110100101100011011100100110 1001011100110010000001 110100011011110110111000100000011011100110111101101101000011 0100001010000011010000 101000001101000010100101001101110101011100100010000001101100 0110010101110011001000 000110100101101101011000010110011101100101011100110010000001 1001000110111101110010 111010010110010101110011000011010000101001010011011101010111 0010001000000110110001 100101011100110010000001100001011100100110110101100101011100 1100100000011001000110 010101110011001000000110011101110101011001010111001001110010 0110100101100101011100 100111001100001101000010100101001101110101011100100010000001 1011000110000100100000 011000110110111101110101011100100110111101101110011011100110 0101001000000110010001 100101011100110010000001110010011011110110100101110011000011 0100001010010010100010 011111101001011000110111001001101001011100110010000001110100 0110111101101110001000 000110111001101111011011010000110100001010000011010000101000 0011010000101001010011 011101010111001000100000011011000110000100100000011010100111 0101011011100110011101 101100011001010010000001100101011101000010000001101100011001 0100100000011001001110 100101110011011001010111001001110100000011010000101001010011 0111010101110010001000 000110110001100101011100110010000001101110011010010110010001 1100110010000001110011 011101010111001000100000011011000110010101110011001000000110 0111011001010110111011 101010011101000111001100001101000010100101001101110101011100 1000100000011011000010 011111101001011000110110100001101111001000000110010001100101 0010000001101101011011 110110111000100000011001010110111001100110011000010110111001 1000110110010100001101 000010100100101000100111111010010110001101110010011010010111 0011001000000111010001 101111011011100010000001101110011011110110110100001101000010 1000001101000010100000 110100001010010100110111010101110010001000000110110001100101 0111001100100000011011 010110010101110010011101100110010101101001011011000110110001 1001010111001100100000 011001000110010101110011001000000110111001110101011010010111 0100011100110000110100 001010010100110111010101110010001000000110110001100101001000 0001110000011000010110 100101101110001000000110001001101100011000010110111001100011 0010000001100100011001 010111001100100000011010100110111101110101011100100110111011 1010010110010101110011 000011010000101001010011011101010111001000100000011011000110 0101011100110010000001 110011011000010110100101110011011011110110111001110011001000 0001100110011010010110 000101101110011000111110100101100101011100110000110100001010 0100101000100111111010 010110001101110010011010010111001100100000011101000110111101 1011100010000001101110 011011110110110100001101000010100000110100001010000011010000 1010010100110111010101 110010001000000111010001101111011101010111001100100000011011 0101100101011100110010 000001100011011010000110100101100110011001100110111101101110 0111001100100000011001 000010011101100001011110100111010101110010000011010000101001 0100110111010101110010 001000000110110000100111111010010111010001100001011011100110 0111001000000111001101 101111011011000110010101101001011011000010000001101101011011 1101101001011100110110 100100001101000010100101001101110101011100100010000001101100 0110010100100000011011 000110000101100011001000000110110001110101011011100110010100 1000000111011001101001 011101100110000101101110011101000110010100001101000010100100 1010001001111110100101 100011011100100110100101110011001000000111010001101111011011 1000100000011011100110 111101101101000011010000101000001101000010100000110100001010 0101001101110101011100 100010000001101100011001010111001100100000011000110110100001 1000010110110101110000 011100110010000001110011011101010111001000100000011011000010 0111011010000110111101 110010011010010111101001101111011011100000110100001010010100 1101110101011100100010 000001101100011001010111001100100000011000010110100101101100 0110010101110011001000 000110010001100101011100110010000001101111011010010111001101 1001010110000101110101 011110000000110100001010010001010111010000100000011100110111 0101011100100010000001 101100011001010010000001101101011011110111010101101100011010 0101101110001000000110 010001100101011100110010000001101111011011010110001001110010 0110010101110011000011 010000101001001010001001111110100101100011011100100110100101 1100110010000001110100 011011110110111000100000011011100110111101101101000011010000 1010000011010000101000 001101000010100101001101110101011100100010000001100011011010 0001100001011100010111 010101100101001000000110001001101111011101010110011001100110 1110100101100101001000 000110010000100111011000010111010101110010011011110111001001 1001010000110100001010 010100110111010101110010001000000110110001100001001000000110 1101011001010111001000 100000011100110111010101110010001000000110110001100101011100 1100100000011000100110 000101110100011001010110000101110101011110000000110100001010 0101001101110101011100 100010000001101100011000010010000001101101011011110110111001 1101000110000101100111 011011100110010100100000011001001110100101101101011001010110 1110011101000110010100 001101000010100100101000100111111010010110001101110010011010 0101110011001000000111 010001101111011011100010000001101110011011110110110100001101 0000101000001101000010 100000110100001010010100110111010101110010001000000110110001 1000010010000001101101 011011110111010101110011011100110110010100100000011001000110 0101011100110010000001 101110011101010110000101100111011001010111001100001101000010 1001010011011101010111

001000100000011011000110010101110011001000000111001101110 1010110010101110101011100

100111001100100000011001000110010100100000011011000010011 1011011110111001001100001 011001110110010100001101000010100101001101110101011100100010 0000011011000110000100 100000011100000110110001110101011010010110010100100000111010 0101110000011000010110

100101110011011100110110010100100000011001010111010000100 0000110011001100001011001

000110010100001101000010100100101000100111111010010110001 1011100100110100101110011

001000000111010001101111011011100010000001101110011011110 1101101000011010000101000

001101000010100000110100001010010100110111010101110010001 0000001101100011001010111

001100100000011001100110111101110010011011010110010101110 0110010000001110011011000

110110100101101110011101000110100101101100011011000110000 1011011100111010001100101

011100110000110100001010010100110111010101110010001000000 1101100011001010111001100

100000011000110110110001101111011000110110100001100101011 1001100100000011001000110

010101110011001000000110001101101111011101010110110001100 1010111010101110010011100

110000110100001010010100110111010101110010001000000110110 0011000010010000001110110

111010010111001001101001011101001110100100100000011100000 1101000011110010111001101

101001011100010111010101100101000011010000101001001010001 0011111101001011000110111

001001101001011100110010000001110100011011110110111000100 0000110111001101111011011 010000110100001010000011010000101000001101000010100101001101 1101010111001000100000 011011000110010101110011001000000111001101100101011011100111 0100011010010110010101

110010011100110010000011101001011101100110010101101001011 0110001101100111010010111

001100001101000010100101001101110101011100100010000001101 1000110010101110011001000

000111001001101111011101010111010001100101011100110010000 0011001001110100101110000

011011000110111101111001111010010110010101110011000011010 0001010010100110111010101

110010001000000110110001100101011100110010000001110000011 0110001100001011000110110

010101110011001000000111000101110101011010010010000001100 1001110100101100010011011

110111001001100100011001010110111001110100000011010000101 0010010100010011111101001

011000110111001001101001011100110010000001110100011011110 1101110001000000110111001

101111011011010000110100001010000011010000101000001101000 0101001010011011101010111

001000100000011011000110000100100000011011000110000101101 1010111000001100101001000

000111000101110101011010010010000001110011001001110110000 1011011000110110001110101

011011010110010100001101000010100101001101110101011100100 0100000011011000110000100

100000011011000110000101101101011100000110010100100000011 1000101110101011010010010

000001110011001001111110100101110100011001010110100101101 1100111010000001101000010

100101001101110101011100100010000001101101011001010111001 1001000000110110101100001 011010010111001101101111011011100111001100100000011100101110 1001011101010110111001 101001011001010111001100001101000010100100101000100111111010 0101100011011100100110

100101110011001000000111010001101111011011100010000001101 1100110111101101101000011 010000101000001101000010100000110100001010010100110111010101 1100100010000001101100 011001010010000001100110011100100111010101101001011101000010 0000011000110110111101

110101011100001110100100100000011001010110111000100000011 0010001100101011101010111

100000001101000010100100010001110101001000000110110101101 0010111001001101111011010

010111001000100000011001010111010000100000011001000110010 1001000000110110101100001

001000000110001101101000011000010110110101100010011100100 1100101000011010000101001

010011011101010111001000100000011011010110111101101110001 0000001101100011010010111

010000100000011000110110111101110001011101010110100101101 1000110110001100101001000

000111011001101001011001000110010100001101000010100100101 0001001111110100101100011

011100100110100101110011001000000111010001101111011011100 0100000011011100110111101

101101000011010000101000001101000010100000110100001010010 1001101110101011100100010 000001101101011011110110111000100000011000110110100001101001 0110010101101110001000 000110011101101111011101010111001001101101011000010110111001 1001000010000001100101 011101000010000001110100011001010110111001100100011100100110 0101000011010000101001

010011011101010111001000100000011100110110010101110011001 0000001101111011100100110

010101101001011011000110110001100101011100110010000001100 1000111001001100101011100

110111001111101001011001010111001100001101000010100101001 1011101010111001000100000

011100110110000100100000011100000110000101110100011101000 1100101001000000110110101

100001011011000110000101100100011100100110111101101001011 1010001100101000011010000

101001001010001001111110100101100011011100100110100101110 0110010000001110100011011

110110111000100000011011100110111101101101000011010000101 0000011010000101000001101

000010100101001101110101011100100010000001101100011001010 0100000011101000111001001

100101011011010111000001101100011010010110111000100000011 0010001100101001000000110

110101100001001000000111000001101111011100100111010001100 1010000110100001010010100

110111010101110010001000000110110001100101011100110010000 0011011110110001001101010

011001010111010001110011001000000110011001100001011011010 1101001011011000110100101

100101011100100111001100001101000010100101001101110101011 1001000100000011011000110

010100100000011001100110110001101111011101000010000001100 1000111010100100000011001

100110010101110101001000000110001011101001011011100110100 1000011010000101001001010

001001111110100101100011011100100110100101110011001000000 1110100011011110110111000

100000011011100110111101101101000011010000101000001101000 0101000001101000010100101

001101110101011100100010000001110100011011110111010101110 1000110010100100000011000

110110100001100001011010010111001000100000011000010110001 1011000110110111101110010

011001001110100101100101000011010000101001010011011101010 1110010001000000110110001

100101001000000110011001110010011011110110111001110100001 0000001100100011001010010

000001101101011001010111001100100000011000010110110101101 0010111001100001101000010

100101001101110101011100100010000001100011011010000110000 1011100010111010101100101

001000000110110101100001011010010110111000100000011100010 1110101011010010010000001

110011011001010010000001110100011001010110111001100100000 0110100001010010010100010

011111101001011000110111001001101001011100110010000001110 1000110111101101110001000

000110111001101111011011010000110100001010000011010000101 0000011010000101001010011

011101010111001000100000011011000110000100100000011101100 1101001011101000111001001

100101001000000110010001100101011100110010000001110011011 1010101110010011100000111

001001101001011100110110010101110011000011010000101001010 0110111010101110010001000

000110110001100101011100110010000001101100111010000111011 0011100100110010101110011

001000000110000101110100011101000110010101101110011101000 1101001011101100110010101

110011000011010000101001000010011010010110010101101110001 0000001100001011101010010

110101100100011001010111001101110011011101010111001100100 0000110010001110101001000

000111001101101001011011000110010101101110011000110110010 1000011010000101001001010

001001111110100101100011011100100110100101110011001000000 1110100011011110110111000

100000011011100110111101101101000011010000101000001101000 0101000001101000010100101

001101110101011100100010000001101101011001010111001100100 0000111001001100101011001

100111010101100111011001010111001100100000011001001110100 1011101000111001001110101

011010010111010001110011000011010000101001010011011101010 1110010001000000110110101

100101011100110010000001110000011010000110000101110010011 0010101110011001000001110

100101100011011100100110111101110101011011001110100101110 0110000110100001010010100

110111010101110010001000000110110001100101011100110010000 0011011010111010101110010

011100110010000001100100011001010010000001101101011011110 1101110001000000110010101

101110011011100111010101101001000011010000101001001010001 0011111101001011000110111

001001101001011100110010000001110100011011110110111000100 0000110111001101111011011

010000110100001010000011010000101000001101000010100101001 1011101010111001000100000

011011000010011101100001011000100111001101100101011011100 1100011011001010010000001

110011011000010110111001110011001000000110010011101001011 1001101101001011100100000

110100001010010100110111010101110010001000000110110001100 0010010000001110011011011

110110110001101001011101000111010101100100011001010010000 0011011100111010101100101

000011010000101001010011011101010111001000100000011011000 1100101011100110010000001 101101011000010111001001100011011010000110010101110011001000 0001100100011001010010 000001101100011000010010000001101101011011110111001001110100 0000110100001010010010 100010011111101001011000110111001001101001011100110010000001 1101000110111101101110

001000000110111001101111011011010000110100001010000011010 0001010000011010000101001

010011011101010111001000100000011011000110000100100000011 1001101100001011011100111

010011101001001000000111001001100101011101100110010101101 1100111010101100101000011 010000101001010011011101010111001000100000011011000110010100 1000000111001001101001 011100110111000101110101011001010010000001100100011010010111 0011011100000110000101

110010011101010000110100001010010100110111010101110010001 0000001101100001001110110 010101110011011100000110111101101001011100100010000001110011 0110000101101110011100 110010000001110011011011110111010101110110011001010110111001 1010010111001000001101

000010100100101000100111111010010110001101110010011010010 1110011001000000111010001 101111011011100010000001101110011011110110110100001101000010 1000001101000010100000 110100001010010001010111010000100000011100000110000101110010 0010000001101100011001

010010000001110000011011110111010101110110011011110110100 1011100100010000001100100

001001110111010101101110001000000110110101101111011101000 0001101000010100100101001

100101001000000111001001100101011000110110111101101101011 0110101100101011011100110 001101100101001000000110110101100001001000000111011001101001 0110010100001101000010 100100101001100101001000000111001101110101011010010111001100 1000000110111011101001

001000000111000001101111011101010111001000100000011101000 1100101001000000110001101 101111011011100110111001100001111011100111010001110010011001 0100001101000010100101 000001101111011101010111001000100000011101000110010100100000 0110111001101111011011

010110110101100101011100100000110100001010000011010000101 0000011010000101001001100 011010010110001001100101011100100111010011101001001011100000 1101000010100000110100 001010000011010000101001010000011000010111010101101100001000 0001000101011011000111

010101100001011100100110010000001101000010100000110100001 0100000110100001010001010

100010101000101010001010100010101000001101000010100100010 1011011100110001101101111 011001001110100100100000011100000110000101110010001000000110 1100011001010010000001 000011011001010110111001110100011100100110010100100000010011 1001100001011101000110

100101101111011011100110000101101100001000000110010001100 1010010000001101100011000

010010000001010010011001010110001101101000011001010111001 0011000110110100001100101

001000000101001101100011011010010110010101101110011101000 1101001011001100110100101 110001011101010110010100100000011001010111010000100000010100 1101101111011100100110 001001101111011011100110111001100101001000000101010101101110 0110100101110110011001

010111001001110011011010010111010011101001001000001110000 0001000000101000001100001

011100100110100101110011001011000010000001000110011100100 1100001011011100110001101 100101001011000010000000110010001100000011001000110001001011 1000001101000010100100 000101110110011001010110001100100000011011000110000100100000 0111000001100101011100

100110110101101001011100110111001101101001011011110110111 0001000000110010001100101 011100110010000011001001011001000110100101110100011010010110 1111011011100111001100 100000011001000110010100100000010011010110100101101110011101 0101101001011101000010 1110

Table 2: File A, ISO8859-1 encoding of the original text, 2358 octets

001101110111101010111100101011110010011100011100000000000 0000100110101001101111111 111001101111001110111100000011000000000000000000000000000000 0000000000000000000110 001000000000000000000000000000000000000000000000000000000000 1101100001001110100101

001001111111100000000010010011010100000011111001110101110 1000000000010011000011010 010010000100011001110001111010100010000100111110010010011010 1111111011111110000110 111110011000001101000100010010011100110111110010000110000001 0001000000000000101110

111111010111101000111001100010001010000000111101111011110 1100000110101011100101001 011001100111010000000111010111001100111111000100001111110000 1000001011001101100110 000001000010101001000001101000011110110000000100000001111010 1011100110111000011001 100110111011001010001110101011001101011010000111011010000110 1000000100000011110010 101101001011100110000011010111000100110100011110011001100010 0001000111100110101010 111110100101111101010110100010111110111010001101011001111000 0011011010011000101101 011101110001000100000011000101001110001111001011010101101000 1011000111111110011100 111001111110011011001000101010011111010010010101000000110001 1100111000001011110111 010110011101100111110110000100011101011110001011100110011011 1010111000110111010000 001111010110000110001111011100001101011001100101101110000011 0010100110101010010111 001101111010001011100101011100011111001101101001111110101110 0100001010111001000111 110111001001111011110111010001110101100101001101011111101001 0001010101010101110000 011001011010100101101100110101101111111101100000000100101000 0100000111111110110010 101011100100111010111011101110101001111001001100011010110111 0100100111000001100011 100110111000111001111100111100010111110110011100110101010100 0101000100001101011100 110100000110001101101100111001110010111010101111010000100101 0001111100010000010111 011011000101101110001010100010110010110101111111100010010001 1111111101010010011101 010110111000101001101001001110110100100111010000101000011011 1110000000110100101101 100101101111000011010001011110000001011111011011011100011001 0100001011010011111110 110000111100011001000101001100011001000010010001000101111010 0011000111011100011101 010010100011111110011000001111101011000011110110100110100100 0100001001111110010101 001010100101100001111101011110011110110010111111100110000110 0000100101000000001100 001000100111010101000111110100101101110110110111011111011011 0101000111100101001110 100111001011011011111011010010100100000111011000001001111101 1001101000010011011101 001011000111101110100111011001010100001010110001010000000100 0101000101101110111011 010101110101100001110101011110001110111101001000001101010011 1110011100110100110001 010111011101010000010101000001001101100011110100101101101100 1011001000100000110110 000001101010000100110001010001110101101010011000010101010010 1111001101001000101000 110011101000111100001000000010111110100011011010110110110100 0000011110001100010001 000011110000000010101011000000011000000000000101001000000100 0101010001100001001110 100100111000000000110001110111100000100100100010110101011000 0001101110010001011111 100110101010011011010110010110001111110110100000011101110101 0101010101110001000100 110110010000011101011110011101000000011010100011110110100010 1001001011001101110010 011000011111010101111001101111101110010110010111011001011000 1011011011010000001000 111101001110000110110011000101100001001111001110101001101101 1000110100000011110010 111011001000001110011101011101100000001000101111001010110011 1000011111000000011011 010111111001101101000011100000111101011101001110000000100010 1111110110100110000000 000001111011010110100010110011111011111101001100011000010011 0111011111011110100100 111111010010000000110010000000011001100001111000100010101000 1100001001101011110100 100000010100110001110110100001001111100110110101101100100110 1110101011011110101101 110110010001001101011111010001100101110010010101101010111000 1110100111111101011010 100100111011010011110010011100001000010011001110001000100100 0010111110111111101010 000011101100011110101011000110111000010111001010001100110001 1011110100000111011101 101101101001001011111111110111100011100100111001101010011001 0010000001100010100110 111101011101110010110000111001111010110011011110101100100001 1101101111001111101101 010110000010011011000000101010110111011110001000000110110000 1100001001110111001000 100000001011111110110111110000111110101110010000001110000010 0111001100011100010110 101101001001010101000100010001110111001101100001100111110011 0011100001000101001101 110001010000111000011001000101011001101000011010101100011001 1111001111101001100010 010011000100111010100101010110000111100010111111111101100011 0101101101101100001001 001111110011111110001010000001110101000001000010101111111110 1011101001010110001110 010001110001100110111100100011111100010001101001000011000110 1101001000001111010000 111010011001010101100100110110111011001111100000111001101100 0101000101110111010100 100111010101110101000000011100011000101011110000001111101011 1111010000111100010111 000010001110011101101011010010011011110010110110011011000001 0010010010010011111010 010001001000001011001101100101000110011100010111101010011111 0000011100011000110111 110000100111001101010111011111110101010001011111000001000101 1001111100110000000001 010100111100000001110011010000000001110001000100011010011110 1010100101001011111101 111100111101111101011001001011101011101100010100111111000110 0010010010010001100101 001111001101100101101111111011100111111000011100010100100111 1001101010000101010001 110001010001011100000110110011000101110001011111001011001100 1001011010001101001101 010010101110100001111010111110110000111011110001001011010101 0101000101100111101101 110110110010110100011110101001010101011110111100011101110001 1000011001110000001100 001011001110111001110111110111110011110111100110101101001001 0011000111011000010010 100010011011000010111011010110001110111110001101000111011011 1110111110001001111111 010001110100110010100010001100010010101001110110010001110010 0111011101110110101110 110010100101000111111000111000111001000101110011101000001111 1100011100010101000111 100100011011101101010001001010011100011100110110011101000111 1011000110000010000011 001111111000011101001111011001000000010000001110110011111110 1001011110110110010001 100100100011111010000000101011000111000010110000000100011110 1101000111000101001000 100100100001011110011011011001100001101011010011100000111001 0000111110000001001011 100011111110101101111010011011011001111100100111111011001010 0001100111100101011100 000100000101011101101000100010001001100010001001000100001011 0011000011000111001000 111110000100000001000010101110101101111100100010001101011101 0101010111000000110010 010111111100000011111111100111011000010001001000000011010011 0100100001100000111101 110000011101011000111000011011111110111011000110000101000011 0010010010000000100000 010101001111011010010111101000001100010000110101011000011000 1111100100000011100011 101110001110101001101101110111011001110000100010100000111100 0001011110000101000111 001101111001110100000011000110010101010001000101011110110011 1100111011001111000100 011000010110000011000111010000100100111100000001100011011010 1010001110000100010101 110100001110010000010011010110100000000110000000110100100010 1001110110110011001001 010000011111010100010011011001101001001011101111100011100000 1100010000010100000101 100010101001011101111001111011010111010110100100100100010111 0111101100110011001001 101000010101100111011000000100011100011010110100101101010010 1000010011100110011010 001010101110000001101100101110001110000000001110111000011000 0001010110111111010110 110111110101100001100011000011100111100101000101011011010010 0000011001000111011110 000011001001100000110011001011011110110100101100000100111111 1101010110110100111011 000111110010000010011001010000010011111101111100011101001110 0001101101111011010011 010100001101011000101001111110001000110100100110011010111101 1011011100001110010101 101010101111111000100100001000110101110101000111000001010001 0011010111001110001001 110000100001100100010101010110111110100111101001101010100001 1101010010101110000000 001000111000011100000101110100100001101010110111111000011000 1010110111010100101101 011100111111100001101111100010000101100110101000111110110110 0100111010000100010111 000011011000000010000101101100011111110001011101101001011111 0000000011100100000100 101001100101110011000110011111100011010011000100111110010011 1011011010011111011000 100100010011100101101000000000000000010000010000000110000000 0000000001000010011000 001111101111000000000000011100001011000000010000000000000001 0010000100100001000000 010000000000001100100010010011011000000000000010000000101000 0000010000101001001100 000000100001111100000000000000000000010100000001000110010000 1010000000000000000000 000000000000000000000000000000000000000000000000000000000000 0000010001000110010000 000001001100000000000110100100000000011000100000000001100101 0000000001110010000000 000111010000000000111010010000000000101110000000000111010000 0000000111100000000000 011101000000000000000000000000000001100100000010000000000000 0000000101000000101000 000001000000000000000011110111111001101011001101100011001111 1111011000000000010001 010100000110000000010000000000100000100000001011010010000001 0000000000000000

Table 3: File B, 7z archive of file A with LZMA2 compression, 1137 octets [0179] For this conversion, the nucleotides are selected among four natural nucleotides: adenine (A), thymine (T), cytosine (C) and guanine (G). The conversion of each digital subsequence into a biooctet consists in converting bits 0 at even positions to nucleotide N1=A, bits 1 at even position to nucleotide N2=T, bits 0 at odd position to nucleotide N3=C and bits 1 at odd position to nucleotide N4=G.

[0180] The size of the longest assembly, called a track, was limited to 1024 biooctets. File B comprises more than 1024 biooctets and will therefore be assembled on more than one track. To be able to rearrange the tracks in the right order, a binary barcode was added, composed of four biooctets, at the beginning of each track. A total of 256 to the power of 4 (4 294 967 296) barcodes are available. The first track (track 0) contains barcode 0 composed of the 4 identical biooctets 0 of sequence “AC AC AC AC” (SEQ ID NO: 1107) followed by the first 1020 biooctets of file B. The second track contains barcode 1, composed of 3 octets 0 of sequence “ACACACAC” followed by one biooctet 1 of sequence “ACACACAG”, followed by the last 117 biooctets of file B. A last special biooctet named EOF B of sequence “CAGTCTGT” is added at the end of track 1 to mark the end of the file (EOF). Therefore Track 0 contains 1024 biooctets and Track 1 contains 122 biooctets.

[0181] To generate the DNA molecules corresponding to the two tracks it is possible for example to perform three golden gate assembly steps to assemble the 1146 biooctets (Fig. 1). At step 1, the biooctets are assembled from two libraries containing all biooctets in blocks of 2 biooctets named BioblockX2. At step 2, blocks containing 32 BioblockX2 and named BioblockX64 are assembled. At step 3, blocks containing 16 BioblockX64 and named BioblockX1024 are assembled.

Results

[0182] Two libraries named ‘library A’ and ‘library B’ and containing all the 256 possible biooctets are constructed. The EOF biooctet EOF B is added to library B, which is therefore composed of 257 biooctets. In the two libraries, each biooctet is surrounded by regions comprising a Bsal cleavage site of 11 nucleotides and is contained in a doublestranded replicative plasmid. The variable region of the Bsal cleavage site, named fusion site, is defined for each library. In library A each biooctet is surrounded by the GTAG fusion site upstream of the biooctet and the TGAC fusion site downstream of the biooctet. In library B, each biooctet is surrounded by the TGAC fusion site upstream of the biooctet and the TCAG fusion site downstream of the biooctet. The composition of libraries A and B are provided in Table 4 and their design is presented in Fig. 2.

Table 4: Sequences of library A and library B bioblocks and their surrounding fusion sites. The fusion sites are bolded.

[0183] The presence of the Bsal cleavage site in the library plasmids allows to capture the 1146 required biooctets surrounded by fusion sites, alternating between library A and library B. The plasmids containing the required biooctets from each library are digested by the restriction enzyme Bsal, thus releasing the 1146 biooctets surrounded by their fusion sites. After capturing the x=l 146 biooctets surrounded by their cleavage sites they are assembled together in a fixed order in three steps.

[0184] At step 1, blocks containing 2 biooctets (BioblockX2) are assembled from the 1146 biooctets surrounded by their fusion sites in double-stranded replicative plasmids. Each plasmid contains two internal Bsal cleavage sites in opposite orientation allowing to release, after Bsal cleavage, the fusion sites GTAG and TCAG upstream and downstream of the BioblockX2 respectively. The fusion sites surrounding each biooctet in libraries A and B allow to assemble biooctets from library A in first position and biooctets of library B in second position. The BioblockX2 are assembled in a set of 32 double-stranded replicative plasmids containing regions surrounding BioblockX2 and comprising a cleavage site for the type Ils restriction enzyme BsmBI (Fig. 3). The variable region of the BsmBI cleavage site is defined for each of the 32 plasmids and define ordered positions for assembly of groups of 32 BioblockX2 at step 2 of the assembly process, thanks to a set of 33 fusion sites (Table 5).

FS1_O=AATA FS1_11=GAAC FS1_22=AGCG

FS1_1=TCAA FS1_12=GCAC FS1_23=ACCT

FS1_2=CTTC FS1_13=CGGC FS1_24=AACA

FS1_3=AGTA FS1_14=CGTA FS1_25=GGCA

FS1_4=ACTG FS1_15=GTAA FS1_26=ACGC

FS1_5=CACA FS1_16=CAAC FS1_27=AATC

FS1_6=CCAG FS1_17=GCTA FS1_28=CGAG

FS1_7=CAAA FS1_18=CCGA FS1_29=TCCA

FS1_8=GACC FS1_19=ACGA FSl_30=CCTA

FS1_9=ACTC FS1_2O=AGAA FS1_31=CTAA

FS1 10=CCAC FS1 21=TAAA FS1 32=GGGA

15

Table 5: Fusion sites surrounding BioblockX2 in the 32 recipient plasmids

[0185] A total of 573 plasmids are assembled at step 1. The 36-nucleotide sequences of the 573 BioblockX2 and their surrounding fusion sites correspond to SEQ ID NO: 1 to SEQ ID NO: 573. The first and last group of 4 nucleotides correspond to the fusion sites flanking each BioblockX2. The groups of 4 nucleotides at positions 5-8, 17-20 and 29-32 correspond to the fusion sites from the bioblocks derived from libraries A and B. [0186] As an example, BioblockX2_0 has the following sequence: z TNGTAGACACACACTGACACACACACTCAGTCz (SEQ ID NO: 1). The fusion sites from the bioblocks derived from libraries A and B are bolded, the fusion sites flanking each BioblockX2 (FS1 X) are italicized.

[0187] At step 2, the x=573 BioblockX2 and their surrounding fusion sites are captured by digestion with the BsmBI restriction enzyme and assembled into BioblockX64 comprising 32 BioblockX2 in double-stranded replicative plasmids. Each plasmid contains two internal BsmBI cleavage sites in opposite orientation allowing to release, after BsmBI cleavage, fusion site FS1 0 and fusion site FS1 32 upstream and downstream of the BioblockX64 respectively. The BioblockX2 are assembled in the correct order thanks to the 33 fusion sites in a set of 16 double-stranded replicative plasmids containing regions surrounding BioblockX64 and comprising a cleavage site for the type Ils restriction enzyme Bsal (Fig. 4). The variable region of the Bsal cleavage site is different for each of the 16 plasmids and define ordered positions for assembly of groups of 16 bioblockX64 at step 3 of the assembly process, thanks to a set of 17 fusion sites (Table 6).

FS2_0=AATA FS2_6=AGCG FS2_12=CTAC

FS2_1=AAGG FS2_7=GCTA FS2_13=GAGA

FS2_2=AAAC FS2_8=GGCA FS2_14=CCAG

FS2_3=TAAA FS2_9=ACCT FS2_15=AGAA

FS2_4=ACGA FS2_10=CGTA FS2 16=GCAC

FS2 5=ACTG FS2 11=AACA

Table 6: Fusion sites surrounding BioblockX64 in the 16 recipient plasmids

[0188] A total of 18 plasmids are assembled at step 2, 17 of them contain 32 BioblockX2, while the last one contains 29 BioblockX2. The sequences of the 18 BioblockX64 and their surrounding fusion sites correspond to SEQ ID NO: 574 to SEQ ID NO: 591.

[0189] At step 3, the x= 18 BioblockX64 and their surrounding fusion sites are captured by digestion with the Bsal restriction enzyme and assembled into Biob lockX 1024 comprising 16 BioblockX64 in double-stranded replicative plasmids. Each plasmid contains two internal Bsal cleavage sites in opposite orientation allowing to release, after Bsal cleavage, fusion site FS2 0 and fusion site FS2 16 (Table 6) upstream and downstream of the BioblockX1024 respectively. The bioblockX64 are assembled in the correct order thanks to the 17 fusion sites (Table 6).

[0190] At step 3, two plasmids corresponding to track 0 and track 1 are assembled (Fig. 5). The sequences of the two BioblockX1024 correspond to SEQ ID NO: 592 and SEQ ID NO: 593. Track 0 comprises 1024 biooctets (four barcoding biooctets and the first 1020 biooctets of file B). Track 1 comprises 122 biooctets (four barcoding biooctets, the last 117 biooctets of file B and the special EOF B biooctet).