BACKGROUND OF THE INVENTION

The invention relates to the enzymatic synthesis of DNA and/or RNA via inkjet printing, and more particularly to such synthesis using a move-stop inkjet printing method.

DNA and RNA polynucleotides are linear polymers of nucleotide monomers or analogues thereof which are capable of specifically binding to other polynucleotides by way of a regular pattern of monomer-to-monomer interactions. Polynucleotides typically range in size from a few monomeric units, e.g. 5 to 40 units when they are usually referred to as “oligonucleotides,” to several thousand monomeric units. Usually, polynucleotides comprise the four natural nucleotides, e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA, linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogues, e.g. including modified bases, sugars, or internucleosidic linkages. Polynucleotides may also comprise one or more of deoxyribonucleotides and one or more of ribonucleotides.

When inkjet printing methods are used in an enzymatic polynucleotide synthesis context, defined quantities of an enzymatic solution can be delivered to precise locations on a substrate at a relatively low cost and with minimal wastage. Indeed, a characteristic of enzymatic nucleic acid synthesis on substrates is that polynucleotide purity, defined as having the correct length and sequence, is strongly correlated with the precision of reagent deposition.

Inkjet modes of printing such as single pass and scanning deposit a single droplet of ink in each location on a substrate, thus creating a spot on this substrate. The sizes of the spots are usually similar for every location of said substrate, and they may depend on various parameters like the size of the nozzles of the printer, the waveform used to eject the ink, ink rheological properties and surface parameters. A nozzle is an orifice in an inkjet print head through which ink is sprayed onto the substrate.

Single pass printing and scanning modes of printing thus offer a limited range of in-flight drop volumes and spot sizes on the substrate. It is moreover difficult, when repeating the printing operation, to deposit successive droplets in the same location of the substrate in a precise manner. Indeed, while adjustments to the waveform and alterations to the ink and surface can result in a level of control over spot size on the substrate and precision, they cannot afford control over spot size with precision. Due to this limitation, single pass printing and scanning modes of printing are not ideal for microarray enzymatic synthesis.

SUMMARY OF THE INVENTION

The present invention addresses the above problems and provides a method of inkjet printing for enzymatically synthesizing nucleic acids that allows precise control over spot size on a substrate at the same time as spotting precision.

For this method, there was a need to adapt the commercially available printhead and its software to be able to print not an image but lines or multiple of droplets. It was devised a set of instruction in the form of a 2D image to overcome the limitation of the commercial software.

In this context, the present invention is directed to a method for synthesizing polynucleotides using a move-stop printer with at least one row of nozzles configured for ejecting ink droplets on required rows of reaction sites of a substrate, these reaction sites having at least one free 3 ’-hydroxyl initiator and/or at least one unprotected elongated fragment, such method comprising:

- a set-up step during which a plurality of sets of instructions is implemented in the movestop printer, each set of instructions controlling the functioning of the move-stop printer for at least one row of reaction sites, each instruction of the sets of instructions controlling the firing of at least one of the nozzles;

- a positioning step during which a given row of reaction sites of the substrate is positioned under the row of nozzles;

- a computing step during which a set of instructions for said given row of reaction sites is read by the move-stop printer, this computing step further comprising a reading substep during which each instruction for said given row of reaction sites is read by the move-stop printer;

- a firing step during which according to the set of instructions, at least one nozzle of the row of nozzles ejects droplets of an ink made of synthesis reagents from one ink cartridge of the move-stop printer associated to this row of nozzles to form a spot on at least one reaction site of the given row of reaction sites of the substrate, the volume of the spot depending on the instructions;

- a synthesis step during which the initiator and/or the unprotected elongated fragment is elongated thanks to the synthesis reagents to form a 3 ’-0 -protected elongated fragment;

- a moving step during which the substrate moves according to a longitudinal direction until at least another row of reaction sites is placed under a row of nozzles, with one reaction site positioned under one nozzle; at least the computing, firing, elongating, and moving steps being repeated until every required row of reaction sites of the substrate has been placed under the nozzles of the movestop printer.

The inkjet printing method according to the invention thus allows the enzymatic synthesis of polynucleotides, for instance fragments or entire biomolecules. Such synthesis is facilitated by the fact that the volume of ink that is to be deposited on a specific reaction site of the substrate can be controlled. The substrate is for instance a planar substrate. An initiator is a polynucleotide having a free 3 ’-hydroxyl and comprises at least one cleavable group. A reaction site corresponds to a location on the planar substrate where ink droplets containing only the initiators can be deposited to form spots, which is where the enzymatic synthesis occurs. Further, as used herein, an “elongation spot” is the spot formed on the surface by the ink droplets containing the synthesis reagents for the synthesis step.

The printer used to implement such process is called a move-stop printer as during the positioning step the planar substrate is moved according to the longitudinal direction to place a row of reaction sites under a row of nozzles and then stopped to fix the position of a reaction site regarding a nozzle of the printer, the nozzles being controlled to fire ink onto the reaction sites in a precise manner, thus reducing risks of imprecision. The planar substrate can then be moved again to place another row of reaction sites under the nozzles, as is the case during the moving step. After each of these positioning step and moving step, the planar substrate may be held stationary for a predetermined period of time. The set-up step allows the programming of the move-stop printer, with the implementation of sets of instructions. Each set of instructions controls the functioning of the move-stop printer for a row of reaction sites, while each instruction of the sets of instructions is dedicated to the control of the opening or the closing of one specific nozzle for this same row of reaction sites. As will be described hereinafter, each row of nozzles may be connected to a different ink cartridge. The method of the invention, here called move-stop printing, involves repeat ejection of droplets at a predetermined frequency and with a predetermined number while the nozzles and substrate are stationary: the ejection of droplets from a row of nozzles aligns with reaction sites, these reaction sites having at least one free 3 '-hydroxyl initiator.

More particularly, the set-up step involves different substeps to prepare the move-stop printer to read its set of instructions. As such, there can be a substep in which nucleic acid sequences, which are written in a text-based format such as FASTA, are parsed by the movestop printer. There can then be another parsing substep, during which the move-stop printer parses positions on the planar substrate where ink must be deposited to form the required nucleic acid sequences, these positions corresponding to the reaction sites. Alternatively, the nucleic acid sequences can be automatically assigned to specific reaction sites.

Another substep in the set-up step is dedicated to the generation of the sets of instructions and their respective instructions. Once it has been done, the set-up step may continue with a substep in which printing coordinates are generated. The generation of printing coordinates may depend on coordinates of the printhead, as well as the position of the planar substrate. There is then a loading substep, during which the sets of instructions are loaded in a print queue of a dedicated interface of the move-stop printer. The order of the sets of instructions in the print queue depends on the printing positions previously generated.

The move-stop printer is then programmed. The planar substrate can thus be correctly positioned, meaning that it moves at a medium speed to a predetermined print position in which required reaction sites are positioned under the nozzles. The move-stop printer receives a trigger, and it can read instructions of a particular set of instructions during a computing step and fire the nozzles accordingly during a firing step. During the computing step, the sets of instructions loaded in the print queue are interpreted by the move-stop printer in communication with electronics which control the firing of the nozzles. Such firing of the nozzles allows the synthesis reagents contained in the ink cartridges to be deposited onto the planar substrate in an appropriate reaction site. The ink ejected by a given row of nozzles comes from a single cartridge. There can be multiple ink cartridges each connected to a given row of nozzles.

The synthesis step is the step of the process which corresponds to the synthesis of the polynucleotide per se. The free 3 ’-hydroxyl initiator, or initiator fragment, is a short oligonucleotide with a free 3 ’ -hydroxyl at its end. It can be an initiator nucleic acid fragment, such as a DNA initiator fragment or an RNA initiator fragment. It is to be elongated to obtain a polynucleotide, such elongation involving the synthesis reactants contained in the ink.

The enzymatic synthesis may be carried out using a variety of reagents, the synthesis reagents, that may contain or consist of reactants, wash solutions, deprotection buffers, enzymes, and the like. The term "synthesis reagent" means any reagent used in a synthesis cycle to couple a monomer, particularly a 3’-O-amino- nucleoside triphosphate, to an initiator or elongated fragment, such as, buffers comprising a template-free polymerase, buffers comprising 3’-O-protected-nucleotide monomers, deprotection (or deblocking) buffers, and the like. In various embodiments, an aldehyde scavenger may be a component of one or more of the synthesis reagents. In some embodiments, an aldehyde scavenger may be added to a reaction mixture as a separate synthesis reagent (without other reactants, wash buffers or enzymes). In some embodiments, an aldehyde scavenger is added to a reaction mixture as a component of a synthesis reagent comprising a template-free polymerase.

The completion of the steps of the method corresponds to a cycle, at the end of which the initiator fragment in a reaction site has been elongated with one nucleotide if the elongation step is a success. This cycle can be repeated as many times as necessary to obtain a polynucleotide of a desired length, for instance a polynucleotide of a predetermined sequence. In other words, the cycle can be repeated until every set of instructions loaded in the print queue has been read.

As an option of the invention, instructions control a number of pulses delivered during the firing of said specific nozzle.

In some embodiments, the number of pulses delivered during the firing of said specific nozzle is in the range of 30 to 600, preferably 40 to 400, more preferably 50 to 200, even more preferably 70 to 170.

In some embodiments, the volume of the spot, which can be either a reaction site or an elongation spot, depends on the number of pulses delivered during this firing.

The volume of synthesis reagents deposited onto the planar substrate in each reaction site can thus be controlled. A way of controlling such volume is to vary the number of pulses delivered while the nozzle is opened, one pulse corresponding to a known volume of ink. Additionally, the volume of synthesis reagents delivered can be controlled by the waveform, such as for instance the flexing of a piezo membrane in the case of a silicon piezoelectric microelectromechanical technology. Both the waveform and the number of pulses may be contingent to the instructions read by the move-stop printer.

In some embodiments, the synthesis reagents comprise a 3’-O-protected nucleoside triphosphate and an elongation enzyme.

The role of the elongation enzyme is to elongate the initiator fragment. Such elongation enzyme can be a template-free polymerase such as a DNA polymerase and/or an RNA polymerase. In alternative embodiments, the 3'-O-protected nucleotides could have tetraphosphates and pentaphosphates as well as alkyl protection on the terminal phosphate. The synthesis reagents may also comprise dinucleotides such as dicaptides.

In some embodiments, the 3’-O-protected nucleoside triphosphate is modified adenine, modified cytosine, modified guanine, modified thymine and/or modified uracil.

These are modified nucleotides, which are nucleotides associated with aminoxy protecting groups so that they are 3’-O-blocked nucleotides. Protection of the nucleotides ensures that the initiator fragment can be elongated with one nucleotide at a time, thus avoiding mistakes in the polynucleotide sequence.

Modified thymine would be used preferentially in DNA synthesis, while modified uracil would be used preferentially to elongate an RNA initiator fragment.

According to an optional characteristic, the modified adenine is contained in a first ink cartridge of the move-stop printer, the modified cytosine is contained in a second ink cartridge, the modified guanine is contained in a third ink cartridge and the modified thymine and/or a modified uracil is contained in a fourth ink cartridge.

The move-stop printer thus comprises at least four ink cartridges each connected to a row of nozzles, so that ink can be flowed from the ink cartridges and delivered to the planar substrate via the nozzles. Here, each cartridge contains different synthesis reagents, and more precisely different nucleotides. Each row of nozzles of the move-stop printer can then be connected to one of these cartridges, so that for a given cycle of the process and for a given row of reaction sites different nucleotides can be deposited on the planar substrate at the same time.

In some embodiments, the method further comprises a deprotecting step during which the 3’-O-protected elongated fragment is deprotected to form an elongated fragment having a free 3 ’-hydroxyl. This deprotecting step corresponds to a deprotection of the modified nucleotides with which the initiator fragment has been elongated. The deprotecting step is a removal of their aminoxy protecting groups.

According to another optional characteristic, the deprotecting step is carried out by a deprotecting buffer contained in an ink cartridge of the move-stop printer.

As for the ink cartridges containing the synthesis reagents, this ink cartridge containing the deprotecting buffer is connected to the nozzles of the move-stop printer. In other embodiments, a separate printhead is used for the deprotection buffer.

Alternatively, the deprotecting step is carried out by a deprotection bath in a deprotection buffer.

Alternatively, the deprotecting step is carried out by spraying a deprotection buffer onto the substrate.

Such deprotection bath may correspond to the planar substrate being immersed in or rinsed with the deprotection buffer. The planar substrate may alternatively be sprayed with the deprotection buffer for a predetermined time. In other embodiments, the deprotecting step is carried out by creating a flow cell around the planar substrate, with deprotection buffer being flown over the planar substrate, thus reducing reagent consumption.

In some embodiments, the deprotection buffer contains specific reagents that chemically react with a protection group and/or protected moiety, such as reducing agents, enzymes for enzymatic cleavage, or the like.

The reducing agents could be nitrous acid.

In some embodiments, the method further comprises a washing step to wash the elongated fragment after the deprotecting step.

This allows the discarding of deprotection buffer which may subsist once the deprotecting step has been carried out. Such washing step may include rinsing the planar substrate in wash buffer, immersing it in a bath of said wash buffer, spraying it with the wash buffer or creating a flow cell within which the wash buffer is flowed.

In some embodiments, the method further comprises a drying step after the deprotecting step or after the washing step. This drying step is carried out in order to decrease the risks of a spot spreading or coalescing with adjacent spots once it has been deposited on the planar substrate. Drying techniques used to this end may be warm air or gas, radiative drying or the like. In order to facilitate the drying, a last wash step with a volatile reagent may be implemented.

In some embodiments, the method further comprises a cleaving step during which the polynucleotides are cleaved from their reaction sites once they have been synthesized.

The cleaving step allows the polynucleotides of interest to be retrieved once they have been synthesized. To this end, a cleavable nucleotide may be inserted at a predetermined location within the original initiator fragment.

In some embodiments, the method further comprises a capping step after the synthesis step, during which the initiator or an elongated fragment with free 3’-O-hydroxyl that failed to be elongated is capped.

Dideoxynucleotide capping causes 3' chain termination; once a cap has been put on a sequence of nucleotides, its elongation is no longer possible.

The capping step is used to avoid a dispersion of lengths at a given reaction site. Indeed, if some fragments fail to be elongated during a given cycle, their capping will ensure they are no longer elongated during following cycles. When all the cycles have been completed, these defective sequences will thus be of a shorter length compared to other polynucleotides of the same reaction site having been elongated at each cycle.

In some embodiments, the capped elongated fragment possesses catchable groups such as biotin, azide or alkyne.

In some embodiments, the initiators are added by other means than inkjet printing on the reaction site.

In the above embodiment, the diameter of a reaction site is between 2 and 10 mm, preferably between 1 and 7 mm.

In other embodiments, the initiators are added directly by the nozzles on the reaction site.

In the above embodiment, the diameter of a reaction site is between 5 and 800 pm, preferably between 6 and 500 pm, more preferably between 7 and 250 pm.

In the embodiments where the initiators are added by the nozzles, the diameter of a reaction site is smaller than the elongation spot so when the elongation droplet is deposited, it covers the whole reaction site. In the above embodiment, the method does not comprise a capping step.

In a particular embodiment, the initiators are added by the nozzle on the reaction site which has a diameter comprised between 80 and 500 pm, more than 70 pulses are delivered at each reaction site, no capping step were used.

In some embodiments, the method further comprises an incubation step.

The incubation step takes place once every row of reactions sites has, been placed under the nozzles, and it corresponds to the reaction time necessary for the elongation to occur. In some embodiments, predetermined incubation periods or reaction times may be in the range of from 30 seconds to 30 minutes, or from 1 min to 30 min, or from 1 min to 15 min, or from 1 min 25 to 10 min, or from 30 sec to 5 min.

In some embodiments, the volume of the droplet is comprised between 3 and 30 pL.

The volume of the droplet may more precisely be comprised between 3 and 10 pL.

In some embodiments, two rows of the reaction sites are configured so that a reaction site of a first row and a reaction site of a second row are aligned parallel to the longitudinal direction which corresponds to the moving direction of the substrate.

The reaction sites are thus aligned on the planar substrate to form a grid pattern, the rows of reaction sites being parallel to each other.

According to a characteristic, each reaction site is distinct and non-overlapping with another reaction site.

Combined with the immobilization of the planar substrate when the nozzles are fired, this characteristic contributes to reducing imprecision and impurity risks.

In some embodiments, a set of instructions corresponds to an image with lines and each instruction of such a set of instructions corresponds to one of the said lines of the image.

In such embodiments, the image can be a barcode whose lines are the instructions for the move-stop printer. This 2D image will give a ID result for spots printed on the support. The barcode is composed of horizontal lines of different length. If there is no line at a certain position, it means that the corresponding nozzle in a given row will not be fired. A vertical position of these lines determines which nozzles in a given row of nozzles will be fired. The length of the lines controls the number of pulses delivered during the firing of the nozzles. In other words, when an image is read the move-stop printer can determine the length of the lines forming the barcode. The longer the lines, the more pulses will be delivered. Each line corresponds to a given nozzle and will result in a droplet or multiple droplets being deposited on a given reaction site to form a spot. The length of the lines determines the volume of synthesis reagents which will be deposited on a given reaction site. The choice of the vertical and horizontal axis is arbitrary and can be inverted with no effect on the result. Also, the line could be read from right to left with no effect on the result.

The length of the line will determine the number of droplets that are fired by the nozzle and so it will determine the final volume of the formed spot.

Other means of digital encoding could be used to encode the printing information. A deck of images can be used to program the move-stop printer, the images then being read one at a time with each image corresponding to a set of instructions for a given row of the planar substrate. As mentioned before, during the set-up step one substep is dedicated to the generation of the sets of instructions and their instructions.

In some embodiments, the polynucleotides are DNA molecules and/or RNA molecules.

Such polynucleotides may refer to either a single stranded form or a double stranded form, as well as RNA/DNA duplexes.

In some embodiments, at least the computing, firing, elongating, and moving steps form a cycle, a volume of a spot formed in a first cycle being smaller than a volume of a spot formed in a subsequent cycle. As a result, the more cycles there are, the bigger the spots get. This is to ensure that an initial reaction site defined during the first cycle is always covered with ink in subsequent cycles.

The invention also intends to cover a move-stop printer, configured with the means to implement a method for synthesizing polynucleotides as previously described. It especially true that this printer should have the means to decode the set of instructions in the form of barcode and use those instructions to obtain synthetized polynucleotides.

In some embodiments, the move-stop printer comprises ink containing dyes, cofactors, aldehyde or ketone scavengers, viscosity modifiers, surfactants, humectants, cosolvents, melting temperature modifiers, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS Other characteristics, details and advantages of the invention will become clearer on reading the following description, on the one hand, and several examples of realisation given as an indication and without limitation with reference to the schematic drawings annexed, on the other hand, on which:

[Fig. 1] is a schematic representation of a move-stop printer for the carrying out of the method for synthesizing polynucleotides according to the invention;

[Fig. 2] is a schematic representation of a set of instructions for the move-stop printer of figure 1 and its printed result;

[Fig. 3] is a schematic representation of the printed result of another set of instructions for the move-stop printer of figure 1;

[Fig. 4] is a schematic representation of a first embodiment of the functioning of the movestop printer of figure 1 ;

[Fig. 5] is a schematic representation of a second embodiment of the functioning of the move-stop printer of figure 1 ;

[Fig. 6] is a schematic representation of a third embodiment of the functioning of the movestop printer of figure 1 ;

[Fig. 7] is a schematic representation of a planar substrate used for the method for synthesizing polynucleotides;

[Fig. 8] is a nozzle of the move-stop printer depositing ink on the planar substrate of figure 7;

[Fig. 9] is another schematic representation of the planar substrate of figure 7;

[Fig. 10] is a diagram of the steps of the method for synthesizing polynucleotides according to the invention.

[Fig. 11 A] is the barcode used in the example for the initiator deposition.

[Fig. 1 IB] is the barcode used in the example for the elongation deposition.

[Fig. 12] is the picture of the result of the initiator deposition in the example using the barcode of figure 11 A.

[Fig.13] is the picture of the result of the elongation deposition in the example using the barcode of figure 1 IB. [Fig.14] is the picture of the electrophoresis gel in the example.

DETAILED DESCRIPTION OF THE INVENTION

The characteristics, variants and different modes of realization of the invention may be associated with each other in various combinations, in so far as they are not incompatible or exclusive with each other. In particular, variants of the invention comprising only a selection of features subsequently described in from the other features described may be imagined, if this selection of features is enough to confer a technical advantage and/or to differentiate the invention from prior art.

Like numbers refer to like elements throughout drawings.

Figure 1 illustrates a move-stop printer 1 according to the invention, such move-stop printer 1 being capable of carrying out a method for enzymatically synthesizing polynucleotides. Such polynucleotides may be biomolecules like DNA molecules and/or RNA molecules and may refer either to single stranded forms or double stranded forms of these molecules. The resulting printing of the move-stop printer 1 can for instance be a microarray.

The move-stop printer 1 comprises a printhead 2 which is equipped with multiple rows of nozzles 4, one of these nozzles 4 being particularly visible on figure 8. The move-stop printer 1 is configured so that ink 6 can be flown from a given ink cartridge to the nozzles 4 of a given row of nozzles 4. The ink 6 used in the move-stop printer 1 contains synthesis reagents which are necessary for an elongation reaction or synthesis step 8 which will be described hereinafter. In addition, the ink 6 may contain viscosity modifiers, humectants, surfactants, aldehyde or ketone scavengers and the like. According to the invention, the synthesis reagents may comprise a 3’-O-protected nucleoside triphosphate as well as an elongation enzyme. This elongation enzyme can for instance be a template-free polymerase. If the molecule to be synthesized is a DNA molecule, the polymerase will be a DNA polymerase, whereas if an RNA molecule is sought the polymerase will be an RNA polymerase. The nucleoside triphosphate contained in the synthesis reagents is protected, which means that it is associated with an aminoxy protecting group so as to block its elongation.

As mentioned before, the ink 6 used in the move-stop printer 1 may be stored in ink cartridges. As such, the number of ink cartridges depends on the 3’-O-protected nucleoside triphosphates to be used for the synthesis step 8. The 3 ’ -O-protected nucleoside triphosphates are modified adenine, modified cytosine, modified guanine, modified thymine and/or modified uracil. Consequently, there can be a first ink cartridge 10 for ink 6 containing modified adenine and the elongation enzyme, a second ink cartridge 12 for ink 6 containing modified cytosine and the elongation enzyme, a third ink cartridge 14 for ink 6 containing modified guanine and the elongation enzyme and a fourth ink cartridge 16 for ink 6 containing modified thymine and/or modified uracil and the elongation enzyme. In particular embodiments and although not shown on the figures, both the modified thymine and the modified uracil may be contained in separate ink cartridges; therefore, there would be five ink cartridges. There could be additional ink cartridges in the move-stop printer, for instance dedicated to fhiorophores for labelling.

The synthesis reagents comprised in the ink 6 are to be deposited on a substrate 18, which is here a planar substrate 18. This planar substrate 18 can be glass, silica, silicon oxide, plastic, or like surfaces, but it may also take place on other surfaces, such as, for example, biological tissues, flexible surfaces such as polyethylene terephthalate (PET), or surface-immobilized cDNAs extracted from tissues. In some embodiments, the planar substrate 18 is multifunctional; it can as such comprise electrodes. As particularly visible on the figures 7 and 9, the planar substrate 18 is of rectangular shape and mainly extends according to a longitudinal direction L from a first longitudinal end 20 to a second longitudinal end 22. The planar substrate 18 substrate may have a surface area in the range of from 1 to 500 cm ² , for instance from 1 to 15 cm ² or from 7 to 20 cm ² .

In the present embodiment, the planar substrate comprises multiple rows 24 of circular reaction sites 26, the rows 24 extending perpendicular to the longitudinal direction L and being successively arranged on the planar substrate 18 according to the longitudinal direction L. The rows 24 are thus parallel one to each other, as particularly visible on figures 2, 7 and 9. The rows 24 are furthermore arranged on the planar substrate 18 so that the reaction sites 26 form a grid pattern or a microarray. As such, a first reaction site 26 A of a first row 24 A and a first reaction site 26B of a second row 24B are aligned parallel to the longitudinal direction L, the first row 24A being the closest to the first longitudinal end 20 of the planar substrate 18. Likewise, a second reaction site 26C of the first row 24A and a second reaction site 26D of the second row 24B are aligned parallel to the longitudinal direction L, and so on for every reaction site 26 of every row 24 of the planar substrate 18.

In addition, in the present embodiment each reaction site 26 is distinct and non-overlapping with another reaction site 26. In other words, the reaction sites 26 are not contiguous with each other and they do not share borders. A surface of each reaction site 26 may be surrounded by a hydrophobic surface on the planar substrate 18, so that each reaction site 26 which may be a hydrophilic surface can be covered in aqueous solution without spreading or coalescing with the aqueous solution of another reaction site 26. Alternatively, the surface between the reaction sites 26 may have no initiator fragment or capped initiator fragments as will be described hereinafter.

A volume of the reactions sites 26 where enzymatic reactions take place may be defined on the planar substrate by wells dug into the planar substrate 18, as illustrated on figure 7. Additionally, these wells may be accompanied by microarray bubbles 28, as shown of figure

9. When the planar substrate 18 is flooded with an aqueous solution, both hydrophobic regions corresponding to spaces between the reaction sites 26 and hydrophilic regions corresponding to these reaction sites 26 are immersed. However, when the aqueous solution drains off, it is not retained by the hydrophobic regions whereas it can be by the hydrophilic regions, thus forming the aforementioned microarray bubbles 28 above each reaction site on the planar substrate 18. These microarray bubbles 28 correspond to a volume, located on the reaction sites 26 of the planar substrate 18, where the DNA and/or RNA synthesis can occur.

The ink 6 is also an aqueous solution. In order to contain this ink 6 and/or other aqueous solutions, the reaction sites 26 may be dug in the planar substrate 18 so as to form circular wells having diameters in the range of from about 20-50 pm, which are excavated from the surface of the planar substrate 18. As mentioned before, such wells are particularly visible on figure 7.

Each reaction site 26 contains at least one free 3 ’-hydroxyl initiator, or initiator fragment. This initiator fragment is a short nucleotide fragment which is to be elongated in order to obtain a polynucleotide, which can be a DNA polynucleotide if the initiator fragment is a DNA fragment or an RNA polynucleotide if the initiator fragment is an RNA fragment.

The method for synthesizing such polynucleotides will now be described referring to figure

10. The method for synthesizing polynucleotides according to the invention comprises a number of steps, the completion of these steps forming a cycle. At the end of a cycle each initiator fragment has been elongated with one nucleotide. The repetition of multiple cycles leads to the synthesis of polynucleotides.

The first step of the method for synthesizing polynucleotides is a set-up step 30; it corresponds to a configuration or programming of the move-stop printer 1. During this set- up step 30, a plurality of sets of instructions 32 are implemented in the move-stop printer 1. Such sets of instructions 32 can for instance be images with lines resembling barcodes, as shown on figure 2. There can be a pile of sets of instructions 32, which are put into a correct order during this set-up step 30. Each set of instructions 32 comprises instructions 34, which when the sets of instructions 32 are images with lines correspond to the lines of the images. Each set of instructions 32 controls the functioning of the move-stop printer 1 for a given row 24 of reaction sites 26, i.e., when the substrate and its reaction sites are immobilized in relation to the nozzles of the move stop printer. Each instruction 34 of a set of instructions 32 controls the opening or the closing of one of the nozzles 4 for this given row 24 of reaction sites 26. Such controls are operated via a controller 35, which can comprise a computer and a software.

The set-up step 30 is followed by a positioning step 36, during which the planar substrate 18 is moved in the move-stop printer 1 so that a row 24 of reaction sites 26 is positioned under the nozzles 4. Such positioning occurs according to the longitudinal direction L. For a first cycle, the row 24 of reaction sites 26 positioned under the nozzles 4 is the first row 24A, which is the row 24 in the vicinity of the first longitudinal end 20 of the planar substrate 18.

When the planar substrate 18 has been adequately positioned relatively to the move-stop printer 1 and is immobile in relation to this move-stop printer, a trigger is sent to the movestop printer 1 and a computing step 38 can occur. Such computing step 38 results in the sets of instructions 32 being read by the move-stop printer 1. The sets of instructions 32 will be read one after the other, according to the order determined during the set-up step 30. As mentioned before, a set of instructions 32, for instance a first image, is linked to a given row 24 of reaction sites 26, namely the first row 24 A for the first cycle. The computing step 38 also comprises a reading substep during which each instruction 34 is read by the move-stop printer 1, each instruction 34 corresponding to the opening of one nozzle straight above a reaction site 26 of the first row 24A for the first cycle. More precisely, a first line on the first image corresponds to the opening of the nozzle straight above the first reaction site 26A of the first row 24A, a second line on the first image corresponds to the opening of the nozzle straight above the second reaction site 26C of the first row 24A, and so on.

This computing step 38 is at least partially concomitant with a firing step 40, during which ink 6 is ejected from the nozzles 4 as illustrated on figure 8. Upon reading of the sets of instructions, the nozzles deliver ink droplets to the planar substrate 18, the droplets being deposited on the reaction sites 26 and more precisely on the microarray bubbles 28 of these reaction sites 26 to form spots 42. As such, synthesis reagents contained in the ink 6 are delivered to reaction sites 26 in the spots 42 generated by the droplets delivered by the nozzles 4. The precision of the ink delivery is helped by the fact that the planar substrate 18 is immobile, as it has been correctly positioned during the positioning step 36.

As the move-stop printer 1 has been programmed during the set-up step 30, a volume of the spots 42 depends on the instructions 34 the move-stop printer 1 has read. These instructions 34 may control a number of pulses delivered during the firing of a specific nozzle 4, as visible on figure 2. The volume of a spot 42 thus depends on the number of pulses delivered during the firing. The longer the nozzles 4 are fired, the more ink 6 and droplets will be deposited onto the planar substrate 18. Likewise, the more pulses are delivered while the nozzles 4 are fired, the more ink 6 and droplets will be deposited on the reaction sites 26, as one pulse corresponds to a known volume of ink 6. In the embodiment of the invention wherein each instruction has the form of a line, the longer the line of an image is, the bigger the corresponding spot 42 will be.

As illustrated on figure 2, there can be a first set of instructions 32A, a second set of instructions 32B and a third set of instructions 32C; here, these sets of instructions 32A, 32B, 32C are respectively a first image, a second image and a third image. The first set of instructions 32A comprises instructions 34A; here, they are lines all of the same length which is a first length. Upon reading of these instructions 34A, droplets will be deposited on the reaction sites 26 of the first row 24A to form spots 42, the droplets having the same size which corresponds to a same number of pulses fired by the nozzles, themselves corresponding to the length of the lines. An image may comprise portions with blanks 33, one of these blanks 33 being represented on figure 2 by dotted lines. A blank 33 equates to no instruction 34A; as such, no corresponding nozzle 4 will be fired, resulting in an absence of spot 42 on a given reaction site 26. Here, as the first set of instructions 32A comprises eight instructions 34A, or lines, droplets will be deposited on eight reaction sites 26 of the first row 24A.

On the second set of instructions 32B, the instructions 34B or lines are also all of the same length, and as such the droplets deposited on the second row 24B of reaction sites 26 will form spots 42 all having the same size. However, as the lines of the second set of instructions 32B are of a shorter length, or second length, compared to the first length of the lines of the first set of instructions 32A, the droplets deposited on the second row 24B will form smaller spots 42 than the ones on the first row 24A. In order to achieve smaller spots 42, the number of pulses the nozzles 4 deliver may be reduced, or the waveform may be varied. Portions where there were blanks 33 on the first set of instructions 32A correspond to portions where there are lines on the second set of instructions 32B; there are thus additional spots 42 on the second row 24B of reaction sites compared to the first row 24A. As illustrated here, there are twelve instructions 34B, or lines, and as such droplets have been deposited to form spots 42 on twelve reaction sites 26 of the second row 24B.

Finally, the third set of instructions 32C comprises instructions 34C, or lines, of different lengths. More precisely, there are lines of the first length, lines of the second length, lines of a third length and lines of a fourth length. Consequently, the droplets deposited on a third row 24C of reaction sites 26 will have four different sizes. As there are eleven instructions 34B, or lines, on the third set of instructions 32C, droplets will be deposited to form spots 42 on eleven reaction sites 26 of the third row 24C.

As is shown in the embodiment of figure 3 with a printhead 2, here shown in transparent mode for illustrative purposes, having a single row of nozzles 4, all the reaction sites 26 of the planar substrate 18 may receive a same amount of ink 6. This is the case when the sets of instructions 32C all comprise a same number of instructions 34C which corresponds to the number of reaction sites 26 of a row 24 of the planar substrate 18, these instructions 34C being of the same length.

The sets of instructions 32C having the same number of instructions 34C as the number of reaction sites 26 of a row 24 results in droplets being deposited in each of the reaction sites 26, whereas the instructions 34C being of the same length results in a same spot 42 being formed in each of these reaction sites 26. It should be noted that these examples of sets of instructions 32 are given by way of illustration and are not restrictive of the invention.

The volume of a droplet is comprised between 3 and 30 pL. The volume of a droplet may more precisely be comprised between 3 and 10 pL.

As mentioned before, the ink 6 contains a 3’-O-protected nucleoside triphosphate and an elongation enzyme. A concentration of the elongation enzyme can be in a range of from 2.0 pM to 200 pM. The concentration of elongation enzyme in a spot 42 may have to be lower than optimal for elongation reactions, as at higher concentrations the enzyme increases the viscosity of the ink such that it negatively impacts droplet formation. To alleviate this issue and obtain a desired concentration, droplets can be repeatedly deposited onto the planar substrate 18 with drying to allow concentration of the enzyme at a reaction site 26. In some embodiments, a plurality of droplets may be deposited on a reaction site 26, for instance by increasing the number of pulses delivered during the firing of the nozzles 4. The number of pulses fired by a given nozzle 4 for a given reaction site 26 may be in the range of 65 to 600, preferably 70 to 400, more preferably 75 to 250.

In addition to viscosity, other key parameters affecting droplet formation are surface tension, liquid density and a diameter of the nozzles 4. The ink 6 can thus be formulated to contain viscosity modifiers, surface tension modifiers, density modifiers and the like, in order to meet the rheological requirements for droplet formation. The diameter of the nozzles 4 can be in the range of from 10 pm to 100 pm.

The role of the aforementioned elongation enzyme contained in the ink 6 is to elongate the initiator fragments present in the reaction sites 26 with the 3’-O-protected nucleoside triphosphates which are also contained in the ink 6; it is the purpose of the synthesis step 8.

As each row of nozzles 4 may be connected to either the first ink cartridge 10, the second ink cartridge 12, the third ink cartridge 14 or the fourth ink cartridge 16, this row of nozzles 4 may deliver either modified adenine, modified cytosine, modified guanine or modified thymine and/or modified uracil.

The elongation enzyme adds one of the modified nucleotides to a free 3 ’-hydroxyl of a 3’- terminal nucleotide of a given initiator fragment, thus resulting in an elongated fragment. Due to the modified nature of the nucleotides which are 3’-O-blocked, for example with an azidomethyl protecting group, only one of them is capable of being linked to the free terminal nucleotide of the initiator fragment. This protection ensures that nucleotides are added to the initiator fragment one at a time for each cycle, therefore avoiding errors in the polynucleotide sequence. In other words, it avoids multiple elongations within a cycle, which would create unwanted homopolymer sequences.

In some embodiments, a capping step 46 may follow the synthesis step 8. During this capping step 46, initiator fragments with free 3’-O-hydroxyl that failed to be elongated are capped with capping agents, which prevent any further elongation of the capped strand. Such capping agents may for example be dideoxynucleoside triphosphates, which may be contained in the synthesis reagents of the ink 6. The capped strands may possess catchable groups such as biotin, azide or alkyne groups, in order to facilitate their separation. Once the preceding steps have been completed, the initiator fragments of a given row 24 of reaction sites 26 have been elongated with one nucleotide. A moving step 47 can then occur, during which the planar substrate 18 moves according to the longitudinal direction L until a next row 24 of reaction sites 26 is placed under the nozzles 4. The computing, firing, elongating and moving steps 38, 40, 8, 47 are then repeated until every required row 24 of the planar substrate 18 has been placed under each row of nozzles 4 of the move-stop printer 1. In other words, once the moving step 47 has been carried out another set of instructions 32 in the form of another image may be read by the move-stop printer 1, the instructions 34 it contains leading to the elongation of the initiator fragments of another row 24 of reaction sites 26 and it is repeated until all set of instructions 32 or images have been read, that is to say until droplets have been deposited to form spots 42 in every row 24 of the planar substrate 18.

The positioning step 36, the firing step 40 and the moving step 47 can be organized differently depending on how the move-stop printer 1 is set up. Three different embodiments will now be described relatively to figures 4 to 6. On these figures, different rows of nozzles 4 are each connected to a different ink cartridge. To differentiate the ink coming from each ink cartridge, spots 42 are shown as symbols, with for instance spots 42 resulting from nozzles 4 connected to the first ink cartridge 10 containing modified adenine being represented as dots, spots 42 resulting from nozzles 4 connected to the second ink cartridge 12 containing modified cytosine being represented as stars, spots 42 resulting from nozzles 4 connected to the third ink cartridge 14 containing modified guanine being represented as triangles, and spots 42 resulting from nozzles 4 connected to the fourth ink cartridge 16 containing modified thymine and/or modified uracil being represented as diamonds.

In figure 4, which is a first embodiment, a single set of instructions 32 is printed before a following moving step 47 occurs. In addition, all the sets of instructions 32 pertaining to certain nozzles 4, which are here nozzles 4 connected to the first ink cartridge 10, are printed before the sets of instructions 32 related to other nozzles 4 can be printed. In this first embodiment, there is thus a first set of instruction 32 printed meaning a first firing step 40 during which ink 6 from the first cartridge 10 is deposited in the form of three spots 42 on the first row 24A of the planar substrate 18. Such three spots 42 of ink 6 from the first cartridge 10 are symbolized as dots. There is then a moving step 47 to position the second row 24B under the nozzles 4 connected to the first ink cartridge 10, and a second firing step 40 which results in ink 6 from the first cartridge 10 being deposited in three spots 42 on the second row 24B of the planar substrate 18. After another moving step 47 to position a third row 24C under the correct nozzles 4, there is a third firing step 40 to deposit ink 6 from the first ink cartridge 10 in a single spot 42 on the third row 24C. These firing and moving steps 40, 47 are repeated until ink 6 from the first cartridge 10 has been delivered to every required reaction site 26. The planar substrate 18 is then positioned in the move-stop printer 1 so that its first row 24 of reaction sites 26 is positioned under nozzles 4 connected to the second ink cartridge 12 containing another nucleotide, and the firing and moving steps 40, 47 may begin again.

In figure 5, which is a second embodiment, there is still a single set of instructions 32 being printed before a following moving step 47 occurs. However, in this embodiment the next nozzles 4 to be fired are not necessarily connected to the same ink cartridge; the next nozzles 4 to be fired are rather determined by the smallest possible move to align a given row 24 of reaction sites 26 of the planar substrate 18 with a row of nozzles 4. As can be seen here, there is a first firing step 40 during which ink from the first cartridge 10 is deposited in the form of three spots 42 on the first row 24 A of the planar substrate 18. There is then a moving step 47 to position the second row 24B under the nozzles 4 connected to the first ink cartridge 10, and a second firing step 40 which results in ink 6 from the first cartridge 10 being deposited in three spots 42 on the second row 24B of the planar substrate 18. Whereas in the first embodiment the following step resulted in ink 6 from the first ink cartridge 10 being deposited in a single spot 42, in this second embodiment the nozzles 4 are fired so that ink 6 from the third ink cartridge 14 is deposited on the first row 24A of the planar substrate 18, because the smallest possible move between the second firing step 40 and the third firing step 40 is to move the first row 24 A from under the nozzles 4 with ink 6 from the second cartridge 12 to the nozzles 4 connected to the third ink cartridge 14. The spot 42 resulting from this firing from nozzles 4 connected to the third ink cartridge 14 is symbolized as a triangle. Subsequently, in the next firing step 40 ink 6 from the second ink cartridge 12 is deposited on the third row 24C of the planar substrate 18 as a single spot 42, which is symbolized as a star. Compared to the first embodiment, this second embodiment thus minimizes the distance moved during the printing. It is optimized for longer planar substrates 18 as it requires fewer moving steps 47, but it might be harder to read for the move-stop printer 1. In figure 6, which is a third embodiment, more than one set of instructions 32 is printed before a following moving step 47 occurs. This results in ink 6 from different ink cartridges being deposited on the planar substrate 18 at the same time. While the first two firing steps 40 are similar to the first and second embodiments, once the planar substrate 18 has been moved for the second time, nozzles 4 connected to the first ink cartridge 10, nozzles 4 connected to the second ink cartridge 12 and nozzles 4 connected to the third ink cartridge 14 are all fired during a same firing step 40, resulting in spots 42 containing different nucleotides being formed on three different rows 24 at the same time. Similarly, for the following firing step 40 which follows another moving step 47, nozzles 4 connected to the first ink cartridge 10, nozzles 4 connected to the third ink cartridge 14 and nozzles connected to the fourth ink cartridge 16 are all fired at once, depositing ink 6 on three different rows 24 in a single firing step 40.

Once all the rows 24 of reaction sites have been placed under the nozzles 4, there is an incubation step 44. This incubation step 44 allows time for the elongation reaction to occur. It can cover predetermined incubation periods or reaction times in the range of, according to different embodiments, from 30 seconds to 30 minutes, more precisely from 1 minute to 30 minutes, even more precisely from 1 minute to 15 minutes, even more precisely from 1 minute to 10 minutes, or even more precisely from 30 seconds to 5 minutes. The incubation step 44 may be conducted at temperatures comprised from 20°C to 65 °C.

The method for synthesizing polynucleotides then comprises a deprotecting step 48, during which the 3 ’ -O-protected elongated fragments are deprotected to form elongated fragments having a free 3 ’-hydroxyl. The deprotecting step 48 is thus a removal of the elongated fragments’ aminoxy protecting groups, so that the elongated fragments may be further elongated in a subsequent cycle. The deprotecting step 48 is carried out by a deprotection buffer which contains specific reagents such as reducing agents, enzymes for enzymatic cleavage, or the like. The reducing agents can for instance be nitrous acid.

Such deprotecting step 48 may be carried out differently according to different embodiments. In some embodiments, the deprotection buffer may be contained in an ink cartridge, as is the case for the 3 ’-O-protected nucleoside triphosphates and the elongation enzyme. The movestop printer 1 may thus comprise a fifth or a sixth ink cartridge dedicated to this deprotection buffer, depending on if there are four or five ink cartridges for the synthesis reactants. In these embodiments, the deprotection buffer can thus be delivered to the planar substrate 18 via the nozzles 4 of the move-stop printer 1. In other embodiments and as illustrated on figure 1, the deprotection buffer is not contained in an ink cartridge and as such will not be delivered by the nozzles 4. In this case, the deprotecting step 48 corresponds to a deprotection bath, the deprotection buffer being stored in a deprotection buffer compartment 50 of the move-stop printer 1. The planar substrate 18 can thus be immersed in or rinsed with the deprotection buffer. Alternatively, the planar substrate 18 could be moved to a wash station to be sprayed with the deprotection buffer for a predetermined amount of time. In that event, the planar substrate 18 would be moved under nozzles connected to a deprotection buffer cartridge to ensure a uniform treatment.

In some embodiments, the deprotecting step 48 may be followed by a second incubating step 52. Typical incubation times for this second incubating step 52 are in the range of from 1 minute to 30 minutes; or in the range of from 3 minutes to 30 minutes; or in the range of from 3 minutes to 15 minutes.

Once the deprotecting step 48 has been carried out, one or several washing steps 54 can occur. During these washing steps 54, the planar substrate 18 is rinsed with a wash buffer contained in a wash buffer compartment 56 of the move-stop printer. This wash buffer compartment 56 is not an ink cartridge and as such the wash buffer is not delivered via the nozzles 4. The planar substrate 18 may be rinsed with the wash buffer or immersed in a bath of it for a predetermined time in order to discard the remaining deprotection buffer. The washing steps 54 may be repeated multiple times until there is no deprotection buffer, enzyme or nucleotide left on the planar substrate 18.

The reagents to be discarded, for instance the deprotection buffer and the wash buffer, may be disposed of in a waste container 58, as visible in figure 1.

Following the washing step 54 or washing steps 54, the method for synthesizing polynucleotides comprises a drying step 60 whose purpose is to decrease the risks of a spot 42 spreading or coalescing with spots 42 contained in adjacent reaction sites 26 once it has been deposited on the planar substrate 18. To this end, drying techniques may include using warm air or gas, radiative drying or the like. The drying may further be facilitated by a washing step 54 involving a volatile solvent.

An inspection system 64 working together with the controller 35 may be added to the movestop printer in order to monitor the synthesis. Such inspection system 64 may comprise a camera which takes images of the planar substrate 18 and an image analysis software which extracts and processes information from the images. Such information may be related to the presence, the absence or the size of the spots 42, as well as where the droplets have been deposited on the planar substrate 18. To this end, the ink 6 may contain dyes in addition to the synthesis reagents, thus facilitating monitoring. The information may be used in real time to optimize synthesis and may be sent to the controller 35 for instance to implement corrective measures.

Once the synthesis of the polynucleotides has been completed, the elongated fragments may be retrieved. As such, the method for synthesizing polynucleotides may further comprise a cleaving step 62 during which the polynucleotides are cleaved from their reaction sites 26.

In alternative embodiments, not shown on the figures, the sets of instructions 32 may not correspond to images and can be mere directions for the positioning and movement of the nozzles 4 as well as their firing.

The present invention thus covers a method for enzymatically synthesizing biomolecules such as DNA and/or RNA using a move-stop printer, a peculiarity of this method residing in the fact that a size of ink spots containing synthesis reactants can be controlled.

Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which the invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

EXAMPLE

The following example is put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed subject matter and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. A synthesis using the method of the application was used using an Epson Precisioncore series printhead and the software used with it was MetPrint from Meteor, droplet volumes are each about 3,5-4 pL. The initiators (5 pM iDNA, 0.65 mM Triton X-100 in 75 v% acetate buffer (0. IM NaOAc 0.5 M NaCl, pH 4.5) and 35 v% DMSO) were deposited on a Poly An 2D azide slide using the instruction from the barcode on figure 11A. 100 pulses of initiators were deposited per reaction sites resulting in 340 pL for a diameter of 100 pm. The resulting reaction sites were displayed on figure 12.

Afterwards for the synthesis step, 125 pulses of elongation ink (0.5 M cacodylic acid (diluted from a 2 M stock at pH 7.4-6.5), 2 mM Tris HCl, 40 mM NaCl, 500-750 pM HEPES, 0.25- 3 mM CoC12, 500-750 pM 3’0NH2-dTTP, 20-25 pM TdT, 50 mM O-benzohydroxylamine hydrochloride, 15-20% DMSO, 0.025-0.05% Tween20 (i.e., 0.2 mM), and 7% (OMe)2PEG500 (i.e., 147 mM)) were deposited using the instruction from the barcode on figure 11B resulting in 425 pL for a diameter of 120 pm. The resulting elongation spots were displayed on figure 13.

After printing the initiators, the slide was incubated 10 minutes at 70% RH. The slide was washed automatically with a buffered proteinase K solution (0.05 mg/mL) for 1 minute, then washed with deprotection buffer (0.7 NaOAc, 1.0 M NaNO2, pH 5,2) for 3 minutes, then rinsed with MQ-water for 1 minute, then dried with compressed air. The slide was repositioned under the printhead and the next cycle was performed. In total, 100 cycles were performed.

After synthesis, elongation spots were cleaved 15 min in PBS (10 pL per 3 mm DNA spot) under UV illumination at 365 nm, spots of the same row were then pooled and 10 pL bromophenol blue stain added before gel electrophoresis. The results are shown in figure 14. On the right, the molecular scale can be found