The invention is related to modified nucleotides and nucleosides and DNA and RNA molecules (oligonucleotides) containing them with increased stability and resistance to nucleolytic degradation. The modified nucleotides and oligonucleotides with increased stability and resistance to nucleolytic degradation are useful in the in vitro selection of aptamers (singlestranded deoxyribonucleic acid (DNA) fragments), used as therapeutic molecules and fundamental blocks of molecular diagnostic tools.

RNA/DNA molecules are susceptible to digestion by nucleolytic enzymes. The use of aptamers with in formulations containing nucleases (actually all biological fluids) is therefore difficult due to nucleic acid degradation by these enzymes. Therefore, the use of aptamers in medical, including therapeutic, applications (drugs, medical devices) or diagnostic applications is difficult or even impossible due to their rapid degradation in the presence of blood, plasma or saliva and thus the loss of function in a product which contains aptamers as functional molecules.

In the publication "Effect of Chemical Modifications on Aptamer Stability in Serum", Kratschmer and Levy, 2017 [1] studies are discussed on the effect of various chemical modifications (2'-deoxy, 2'-hydroxy, 2'-fluoro and 2'-O-methyl) on the stability of a control oligonucleotide sequence after incubation in a frozen human serum and fresh murine and human serum In addition, the effect of 3'-3' inverted thymidine (3'-3' dT) is presented. The authors note that the stability of fYrR (2'-fluoro RNA) is similar to that of unmodified DNA (2'- deoxy). The incorporation of 3'-3' dT had a small only effect on stability in serum. In one case, the incorporation of 3'-3' dT into a DNA molecule made it more stable than its fYrR equivalent. Fully modified oligonucleotides (100% 2-O-methyl or 2'-O-methyl A, C and U in combination with 2'-fluoro G, known as fGmH) had by far the longest half-life. The sequences showed low degradation in human serum even after extended incubation. Review papers "Aptamers Chemistry: Chemical Modifications and Conjugation Strategies", Odeh et al., 2019 [2] and "From selection hits to clinical leads: progress in aptamer discovery", Maier and Levy, 2019 [3] discuss various strategies for modifying aptamers, having a favorable effect on the stability of molecules toward nucleases. As well as the chemical modifications of the sugar ring discussed above, use of LNA (locked nucleic acid) was shown, an analog in which ribose molecules have an additional bond which links carbon 4' and oxygen 2' (thus locking ribose in the 3'-endo- conformation) and 2'-amino substitution and 2'-deoxy-2'-fluoro- modified arabinonucleotide (2'F-ANA), which was successfully used in a thrombin-binding aptamer to significantly increase its structural stability and nucleolytic resistance [4] , Other strategies for the stabilization of RNA/DNA oligonucleotides are based on the protection of the 3' and/or 5' end of the molecule by adding a protective group. As well as the inverted thymidine, biotin may also have this function, and its stabilizing effect has been confirmed for the thrombin-binding aptamer [5] and an SARS-CoV helicase-binding aptamer [6], A polymer, polyethylene glycol (PEG) with a mass of 20-40 kDa, is also widely used for the stabilization of the 5' end and increasing clearance for oligonucleotides. In addition, the phosphodiester bond in the main chain is replaced with a phosphothioester bond in RNA and DNA structures to improve stability [7], The review papers listed several other methods for the stabilization of oligonucleotide molecules, but a technical issue remains that most of the modifications can be introduced only after the selection process, because RNA and DNA polymerases incorporate structurally modified nucleotides to a limited extent, which significantly reduces library diversity during selection. Even though literature reports are available concerning selection using modified nucleotides in the region of the sugar ring, phosphodiester bond and nitrogenous base [8-10], preparation of an oligonucleotide molecule with better stability parameters toward nucleases still remains a multistage and labor-intensive process, typically performed after selection. This results in a number of problems, because a modification incorporated after obtaining the molecule which binds the molecular target may have a negative effect on its binding parameters and requires chemical synthesis of multiple variants modified at different positions, which leads to high cost of the preparation of the final stable oligonucleotide molecule.

In the international patent application PCT W02016050850A1, Tolle et al., 2014 [11] disclosed a method for the preparation of an aptamer, including the following steps: preparation of a nucleic acid mixture, containing at least one nucleotide, which is modified so that it contains functionalization introduced in a click reaction, contacting the mixture with a target sample; separation of bound nucleic acids which bind the target sample; amplification of the bound nucleic acids to generate a population of nucleic acids that bind the target sample, thus producing the aptamer. The click reaction is performed in an oligonucleotide library.

The identification of aptamers against specific molecular targets remains a challenge. The success rate of primary selection reported in the literature is lower than 30% [12], Certain molecular targets are resistant to the selection process and, therefore, the identification of aptamers targeting the proteins is very difficult if not impossible using natural oligonucleotide libraries.

The limited chemical diversity of oligonucleotide libraries is considered the primary cause of the low success rate in selection. Position 5 of deoxyuridine is used as a convenient addition site of various functional groups, such as alkyl or aromatic chains. Such modifications have an enormous effect on the mode of nucleic acid ligand interactions with molecular targets. The authors of "Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents", Rohloff et al., 2014 [13] achieved a success rate of selection greater than 80% through chemical modifications of aptamers which were less polar and more hydrophobic compared to conventional oligonucleotides.

A method for the synthesis and purification of a nucleotide, a modified nucleotide, a DNA molecule and an oligonucleotide library containing the modified nucleotide and use of the oligonucleotide library is disclosed in Polish patent PL 239946, Jurek et al., 2015 [14],

The technical problem in the present invention is to propose modified nucleotides so that they may be used directly in the aptamer selection process by their incorporation into nucleic acids using enzymatic methods with high yield. Therefore, it will be possible to obtain aptamers directly after the selection procedure with better stability parameters against nucleases than aptamers based on natural nucleotides.

The first object of the invention is a method for the synthesis and purification of a nucleoside and/or a nucleotide, being a mono-, di- or triphosphate, characterized in that a Huisgen copper-catalyzed alkyne-azide cycloaddition reaction is performed of a compound of general formula la or with a compound of general formula lb with a compound of general formula II, wherein in substituent F for n=0 R3 is selected from a group including: a primary amine substituent, a methoxy substituent or a hydroxy substituent, a trifluoromethyl substituent or for n=l substituent R3 is selected from a group including: a hydrogen atom, a fluorine atom, a methyl substituent or a vinyl substituent, or with a compound of general formula III or with a compound of general formula IV or with a compound of general formula V or with a compound of general formula VI or with a compound of general formula VII, wherein the reaction mixture contains triethylamine-acetic acid buffer, sodium ascorbate and DMSO, and the reaction is conducted for a period of between 1 and 6 hours at 43°C, and the reaction product is purified using reverse-phase chromatography.

The second object of the invention is a modified nucleoside and/or a nucleotide, being a mono-, di- or triphosphate, containing as the nitrogenous base a cytosine or uracil derivative which, in the 5' position of the heterocyclic ring has a 1,2,3-triazole group or an alkane or an alkyne chain, containing a terminal 1,2,3-triazole group, and a substituent substituted at this 1,2,3-triazole group in position 1', being a derivative of one of the group of compounds with general formulas from II to VII, and the modified nucleoside and/or nucleotide is of general formula la or lb, wherein in substituent R4 for n=0 R3 is selected from a group including: a primary amine substituent, a methoxy substituent or a hydroxy substituent, a trifluoromethyl substituent or for n=l substituent R3 is selected from a group including: a hydrogen atom, a fluorine atom, a methyl substituent or a vinyl substituent, except for substituent R4 of formulas VIII and IX.

The third object of the invention is a DNA molecule containing a single-or double-stranded oligonucleotide chain, characterized in that it contains one or more modified nucleotides in one or more positions of the sequence of any or both strands, as defined in the first object of the invention. Therefore, the molecule has increased, compared with the molecule not containing modified nucleotides, stability and resistance to nucleolytic degradation in the presence of physiological fluids.

The fourth object of the invention is an oligonucleotide library having in the sequence a region with any sequence at least 10 nucleotides in length and two regions flanking it with fixed sequences at least 10 nucleotides in length or without the flanking regions, characterized in that it contains one or more modified nucleotides in one or more positions of the sequence, as defined in the second object of the invention. Therefore, the library and the oligonucleotides (aptamers) derived therefrom have increased, compared with the molecules/libraries not containing modified nucleotides, stability and resistance to nucleolytic degradation in the presence of physiological fluids.

A further object of the invention is the use of the oligonucleotide library, as defined in the fourth object of the invention, for the preparation of aptamers using SELEX and derived technologies.

The embodiments of the invention are shown in the drawings, wherein:

Fig. 1 - Chromatogram of the compound of formula 37, Fig. 2 - Mass spectrum of the compound of formula 37, Fig. 3 - Chromatogram the compound of formula 22, Fig. 4 - Mass spectrum of the compound of formula 22, Figure 5 - Gel after PER reaction for oligonucleotide Apt-8.11-38, Fig. 6 - Gel after PER reaction for LibP.CLB, Fig. 7 - Gel after the stability test in human serum (a) and rat serum (b) for single-stranded oligonucleotide Apt-8.11-38, Fig. 8 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with natural nucleotides, Fig. 9 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 32, Fig. 10 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 33, Fig. 11 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 34, Fig. 12 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 35, Fig. 13 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 36, Fig. 14 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 37, Fig. 15 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 38, Fig. 16 - Gel after the stability test in human serum (a) and rat serum (b) for a singlestranded library with a modified nucleotide of formula 39, Fig. 17 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 40, Fig. 18 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 41, Fig. 19 - Gel after the stability test in human serum (a) and rat serum (b) for a single-strand library with a modified nucleotide of formula 42, Fig. 20 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 43, Fig. 21 - Gel afterthe stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 44, Fig. 22 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 45, Fig. 23 - Gel after the stability test in human serum (a) and rat serum (b) for a single-stranded library with a modified nucleotide of formula 46, Fig. 24 - Stability chart in human serum and rat serum for single-stranded oligonucleotide Apt-8.11-38, Fig. 25 - Stability chart in human serum and rat serum for a single-stranded library with natural nucleotides, Fig. 26 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 32, Fig. 27 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 33, Fig. 28 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 34, Fig. 29 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 35, Fig. 30 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 36, Fig. 31 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 37, Fig. 32 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 38, Fig. 33 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 39, Fig. 34 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 40, Fig. 35 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 41, Fig. 36 - Stability chart in human serum and rat serum for a singlestranded library with a modified nucleotide of formula 42, Fig. 37 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 43, Fig. 38 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 44, Fig. 39 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 45, Fig. 40 - Stability chart in human serum and rat serum for a single-stranded library with a modified nucleotide of formula 46.

Example 1 Synthesis and purification of 5-EdUTP nucleotide (5-ethinyl-2'-deoxyuridine- triphosphate) modified with l-(azidomethyl)-4-chlorobenzene (formula 37)

The synthesis reaction of the modified nucleotide of formula 37 was performed in a volume of 800 pL. To 230 pL water 80 pL of 500 mM TEAA buffer (triethylamine-acetic acid, pH 7.0) and 400 pL DMSO was added. Subsequently 4 pL of 100 mM 5-EdUTP (5-ethinyl 2'- deoxyuridine triphosphate - formula la) solution and 200 mM l-(azidomethyl)-4- chlorobenzene (formula 7) (1.0 pmol, 3 molar equivalents of 5-EdUTP) dissolved in DMSO was added. To the mixture, 40 pL of a previously prepared Cu-TBTA mixture (10 mM CuSO4, 25 mM TBTA (tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine), dissolved in a mixture of 80% DMSO, 20% tert-butanol) was added. All the ingredients were stirred and subsequently 40 pL of 200 mM sodium ascorbate was added, and the reaction was initiated by reducing copper to oxidation state I. The test tube was tightly sealed. The reaction was conducted in a sea led test tube for 3 hours at 43°C with shaking. Additional 2 pL of 200 mM sodium ascorbate was added after 1.5 h to maintain catalytically reactive Cu(l) ions during the reaction. The product was purified using reverse-phase chromatography with 100 mM TEAA buffer and acetonitrile as the mobile phase (chromatographic system: 1260 Infinity II Bio-Inert LC System from Agilent, column ZORBAX 300SB-C18, 3.5 pm, 4.6 x 150 mm from Agilent). The chromatogram of the analytical sample is shown in Figure 1. Figure 2 shows a corresponding mass spectrum. Collected fractions in preparative samples containing the purified nucleotide of formula 37 were pooled, initially evaporated (CentriVap Benchtop Centrifugal Vacuum Concentrator from Labconco) and freeze-dried (Lyophiliser Alpha 1-4 LSCpIus from Martin Christ). Subsequently, samples for quality control were suspended in a known volume of 50% MeCN/MQ and subjected to MS-TOF mass analysis (Xevo G2-XS QTof mass spectrometer from Waters) in the negative electrospray ionization mode: measured mass: 658.0366 m/z, theoretical mass: 659.76 g/mol.

This procedure may also be used to prepare respective derivatives containing a cytosine derivative as the base. The reactions for the preparation of other nucleotides are conducted for a period of between 1 and 6 hours. Shorter or longer times have no significant effect on yield.

Example 2 Synthesis and purification of modified nucleotides of formulas 32-46

Synthesis reactions of nucleotides of formulas 32-46 were performed similarly as that of the compound of formula 37 (Example 1), using analogs of the compound defined in formula la, wherein substituent R2 was an ethinyl substituent reacted with respective azides of formulas 2-16. Table 1 shows masses of the nucleotides defined in formulas 32-46, prepared according to the aforementioned process 2.

Table 1. Mass analysis of products of formulas 32-46.

Example 3. Synthesis and purification of 5-Edll nucleoside (5-ethinyl-2'-deoxyuridine) modified with l-(azidomethyl)-4-chlorobenzene (formula 22) The synthesis reaction of the modified nucleoside of formula 22 was performed in a volume of 800 pL. To 226 pL water 80 pL of 500 mM TEAA buffer (triethylamine-acetic acid, pH 7.0) and 400 pL DMSO was added. Subsequently 8 pL of 100 mM 5-EdU (5-ethinyl 2' -deoxyuridine - formula la) solution and 200 mM l-(azidomethyl)-4-chlorobenzene (formula 7) (1.0 pmol, 1.5 molar equivalents of 5-EdU) dissolved in DMSO was added. To the mixture, 40 pL of a previously prepared Cu-TBTA mixture (10 mM CuSO4, 25 mM TBTA (tris[(l-benzyl-lH-l,2,3- triazol-4-yl)methyl]amine), dissolved in a mixture of 80% DMSO, 20% tert-butanol) was added. All the ingredients were stirred and subsequently 40 pL of 200 mM sodium ascorbate was added, and the reaction was initiated by reducing copper to oxidation state I. The test tube was tightly sealed. The reaction was conducted in a sealed test tube for 3 hours at 43°C with shaking. Additional 2 pL of 200 mM sodium ascorbate was added after 1.5 h to maintain catalytically reactive Cu(l) ions during the reaction. The product was purified using reversephase chromatography with 100 mM TEAA buffer and acetonitrile as the mobile phase (chromatographic system: 1260 Infinity II Bio-Inert LC System from Agilent, column ZORBAX 300SB-C18, 3.5 pm, 4.6 x 150 mm from Agilent). The chromatogram of the analytical sample is shown in Figure 3. Figure 4 shows a corresponding mass spectrum. Collected fractions in preparative samples containing the purified nucleoside of formula 22 were pooled, initially evaporated (CentriVap Benchtop Centrifugal Vacuum Concentrator from Labconco) and freeze-dried (Lyophiliser Alpha 1-4 LSCpIus from Martin Christ). Subsequently, samples for quality control were suspended in a known volume of 50% MeCN/MQ and subjected to MS- TOF mass analysis (Xevo G2-XS QTof mass spectrometer from Waters) in the negative electrospray ionization mode: measured mass: 417.9810 m/z, theoretical mass: 419.76 g/mol.

This procedure may also be used to prepare respective derivatives containing a cytosine derivative as the base. The reactions for the preparation of other nucleosides are conducted for a period of between 1 and 6 hours. Shorter or longer times have no significant effect on yield.

Example 4 Synthesis and purification of modified nucleosides of formulas 17-31

Synthesis reactions of nucleosides of formulas 17-31 were performed similarly as that of the compound of formula 22 (Example 3), using analogs of the compound defined in formula la, wherein substituent R2 was respective azides of formulas 2-16. Table 2 shows masses of the example nucleosides defined in formulas 32-46, prepared according to the aforementioned process 3. However, it will be obvious for a person skilled in the art that nucleosides not listed in Table 2 can be prepared and purified identically as the nucleoside of formula 22.

Table 2. Mass analysis of products of formulas 18, 20, 22, 24, 25, 29, 31.

Example 5 Enzymatic synthesis of single-stranded DNA using a modified nucleotide (an oligonucleotide with a specific sequence with modified nucleotides)

Enzymatic synthesis of an oligonucleotide containing in its sequence modified nucleotides of formula 37 was performed in a volume of 1000 pL using single-strand template Apt-8.11-38. rc (Sequence 1) and NH2-C12_W_ Apt-8.11-20 (Sequence 2) in a PER (Primer Extension Reaction) process. A reaction mixture was prepared, containing NH2-C12_W_ Apt-8.11-20 in a concentration of 4 pM, polymerase 125 U, lx concentrated buffer and 3 mM magnesium supplied by the polymerase manufacturer, nuclease-free water, nucleotide triphosphates: dATP, dCTP and dGTP, each at a concentration of 500 pM and a modified nucleotide of formula 37 diluted 40x (prepared as in Example 2). Negative control (NC sample) did not contain the template, and the test sample contained Apt-8.11-38. rc at a concentration of 1 pM. Reaction mixtures were incubated for eight minutes at 95°C, seven minutes at 56°C and two minutes at 72°C. After the reaction was completed, the samples were subjected to electrophoresis in 10% denaturing (7.8 M urea) polyacrylamide gel (acrylamide 19:15 bis-acrylamide) heated to 56°C, at a constant voltage of 300 V. The gel was visualized using a SybrSafe dye (Thermo Scientific) - Figure 5. A single-stranded oligonucleotide containing the modified nucleotide of formula 37 was obtained by separating the strands of the double-strand product in 10% denaturing (7.8 M urea) polyacrylamide gel (acrylamide 19:1 bis-acrylamide) heated to 56°C, at a constant voltage of 300 V. The gel was visualized using a SybrSafe dye and subsequently bands corresponding to the modified strand were cut out and purified.

Example 6 Enzymatic synthesis of a single-stranded DNA library containing modified nucleotides

Synthesis of a single-strand DNA library containing respective modified nucleotides of formulas 32-46 or four natural nucleotides (adenosine-5'-triphosphate, guanosine-5'- triphosphate, cytidine-5'-triphosphate, thymidine-5'-triphosphate) was performed using the PER method similarly as in Example 5 with modifications: the LibP library (Sequence 3) was used as the template. LibP_Fwd (Sequence 4) was used as the primer. After the reaction ended, the samples were subjected to gel electrophoresis and visualized as in Example 5 - Figure 6.

Example 7 Stability of the single-stranded oligonucleotide containing the modified nucleotide of formula 37

A stability test of single-stranded oligonucleotide Apt-8.11-38 (Sequence 5) containing the modified nucleotide of formula 37 was performed in human and rat serum. Therefore, 73 pL of serum heated to 37°C and 80 pmol of oligonucleotide (~7 pL, concentration: ~11 pM) was pooled, stirred and subsequently aliquoted into separate test tubes in equal volumes of 10 pL. Samples incubated at 37°C were collected in the following timepoints: Oh, lh, 4h, 8h, 12h, 24h. After a specific time elapsed, the whole test volume was transferred into a test tube containing 2 pL of 500 mM EDTA. Subsequently, the samples were denatured at 95°C, 10 min and centrifuged at 20,000 ref, 10 min at room temperature. 6 pL of supernatant containing the oligonucleotide was collected from the precipitate of serum proteins. Part of the sample was subjected to electrophoresis in 10% polyacrylamide gel (acrylamide 19:15 bis-acrylamide) in denaturing conditions (7.8 M urea) heated to 56°C, at a constant voltage of 300 V. The gel was visualized using a SybrSafe dye (Thermo Scientific) - Figure 7a (human serum) and 7b (rat serum). Densitometric analysis using volumetric tools available in Image Lab 6.0.1 was performed. The analytical results are shown in Figure 25. Example 8 Stability of the single-stranded library containing the modified nucleotide of formulas 32-46

A stability test of the DNA library containing the modified nucleotide of formulas 32-46 or a natural nucleotide was performed similarly as in Example 7. The stability test result for a specific modification was each time shown in pairs of Figures 8-23, wherein Figure a) was the result in human serum, and Figure b) was the result in rat serum. Densitometric analysis using volumetric tools available in Image Lab 6.0.1 was performed. The analytical results are shown in Figures 26-41.

References

[1] Kratschmer C, Levy M. Effect of Chemical Modifications on Aptamer Stability in Serum. Nucleic Acid Ther. 2017 Dec;27(6):335-344. doi: 10.1089/nat.2017.0680.

[2] Odeh F, Nsairat H, Alshaer W, Ismail MA, Esawi E, Qaqish B, Bawab AA, Ismail SL Aptamers Chemistry: Chemical Modifications and Conjugation Strategies. Molecules. 2019 Dec 18;25(1):3. doi: 10.3390/molecules25010003.

[3] Maier KE, Levy M. From selection hits to clinical leads: progress in aptamer discovery. Mol Ther Methods Clin Dev. 2016 Apr 6;5:16014. doi: 10.1038/mtm.2016.14.

[4] Peng CG, Damha MJ. G-quadruplex induced stabilization by 2'-deoxy-2'-fluoro-D- arabinonucleic acids (2'F-ANA). Nucleic Acids Res. 2007;35(15):4977-88. doi: 10.1093/nar/gkm520.

[5] Dougan H, Lyster DM, Vo CV, Stafford A, Weitz JI, Hobbs JB. Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood. Nucl Med Biol. 2000 Apr;27(3):289- 97. doi: 10.1016/s0969-8051(99)00103-l.

[6] Shum KT, Tanner JA. Differential inhibitory activities and stabilisation of DNA aptamers against the SARS coronavirus helicase. Chembiochem. 2008 Dec 15;9(18):3037-45. doi: 10.1002/cbic.200800491.

[7] Diafa S, Hollenstein M. Generation of Aptamers with an Expanded Chemical Repertoire.

Molecules. 2015 Sep 14;20(9):16643-71. doi: 10.3390/molecules200916643. [8] Cheng C, Chen YH, Lennox KA, Behlke MA, Davidson BL. In vivo SELEX for Identification of Brain-penetrating Aptamers. Mol Ther Nucleic Acids. 2013 Jan 8;2(l):e67. doi: 10.1038/mtna.2012.59.

[9] Mann AP, Somasunderam A, Nieves-Alicea R, Li X, Hu A, Sood AK, Ferrari M, Gorenstein DG, Tanaka T. Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting. PLoS One. 2010 Sep 30;5(9):el3050. doi:

10.1371/journal.pone.0013050.

[10] Vaught JD, Bock C, Carter J, Fitzwater T, Otis M, Schneider D, Rolando J, Waugh S, Wilcox SK, Eaton BE. Expanding the chemistry of DNA for in vitro selection. J Am Chem Soc. 2010 Mar 31;132(12):4141-51. doi: 10.1021/ja908035g.

[11] Tolle F, Friedrich F, Heckel A, Mayer G. A method of identifying or producing an aptamer. PCT W02016050850A1. 2014.

[12] Famulok M, Mayer G. Aptamers and SELEX in Chemistry & Biology. Chem Biol. 2014 Sep 18;21(9):1055-8. doi: 10.1016/j.chembiol.2014.08.003.

[13] Rohloff JC, Gelinas AD, Jarvis TC, Ochsner UA, Schneider DJ, Gold L, Janjic N. Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents. Mol Ther Nucleic Acids. 2014 Oct 7;3(10):e201. doi: 10.1038/mtna.2014.49.

[14] Jurek P, Jeleri F, Mazurek M, Jakimowicz P. Sposob syntezy i oczyszczania nukleotydu, zmodyfikowany nukleotyd, czqsteczka DNA i biblioteka oligonukleotydow zawierajqce zmodyfikowany nukleotyd oraz zastosowanie biblioteki oligonukleotydow. PL 239946. 2015.

Sequence list

Sequence 1: Apt-8.11-38. rc

5'-TGCTAACCTACAGGCAGACCAGTCGTTGAGGTAAGAGCAAAAAAAAAAAAAAAA AAAAAAA-3'

Sequence 2: NH2-C12_W_Apt-8.11-20 5'- [NH2-C12] CAACGACTGG -3'

Sequence 3: LibP

5'- GCT TGA ATG CAT ACC CTG-40N-GTA CTG CTG CCT GTC TAT-3'

Sequence 4: Fwd_LibP

5'-CAACGCTATCGGTCGTAG-3' Sequence 5: Apt-8.11-38

5'[NH2-C12] CAACGACTGGU ^CLB CU ^CLB GCCU ^CLB GU ^CLB AGGU ^CLB U ^CLB AGCA 3'