Purification of DNA-Conjugate Products

Abstract

This invention relates to the purification of nucleic acid conjugates, for example for use in DNA encoded chemical libraries. Reaction members that comprise nucleic acid conjugate reaction products and nucleic acid or nucleic acid conjugate reactants comprising a first reactive group are contacted with a capping molecule comprising a capture group. The capping molecule reacts with the first reactive group to form a covalent bond, thereby attaching the capture group to nucleic acid or nucleic acid conjugate reactants in reaction members. The nucleic acid or nucleic acid conjugate reactants are then removed from the reaction members using a binding member that binds the capture group, thereby producing a purified population of nucleic acid conjugate products. Methods of producing purified populations of nucleic acid conjugate products and nucleic acid chemical libraries are provided along with chemical libraries and kits.

Claims

1. A method of producing a purified population of nucleic acid conjugate reaction products comprising; providing reaction members that comprise nucleic acid conjugate reaction products and nucleic acid or nucleic acid conjugate reactants, wherein said reactants comprise a first reactive group, contacting the reaction members with a capping molecule comprising a capture group, wherein the capping molecule reacts with the first reactive group to form a covalent bond, thereby attaching the capture group to nucleic acid or nucleic acid conjugate reactants in reaction members, removing the nucleic acid or nucleic acid conjugate reactants from the reaction members using a binding member that binds the capture group, thereby producing a purified population of nucleic acid conjugate products.

2. A method according to claim 1 wherein the reaction members are provided by a method comprising; reacting a population of nucleic acid or nucleic acid conjugate reactants having a first reactive group with a population of chemical moieties that react with the first reactive group to produce reaction members comprising nucleic acid conjugate products that contain the chemical moiety and unreacted nucleic acid or nucleic acid conjugate reactants.

3. A method according to claim 1 wherein the reaction members comprise nucleic acid strands having a first reactive group at a terminal of the nucleic acid strands.

4. (canceled)

5. A method according to claim 1 wherein the reaction members comprise nucleic acid conjugate reactants having a first reactive group.

6. A method according to claim 5 wherein the nucleic acid conjugate reactants comprise a nucleic acid strand conjugated to at least one chemical moiety and wherein the conjugated chemical moiety comprises the first reactive group.

7. (canceled)

8. A method according to claim 6 wherein the nucleic acid conjugate reactants comprise a nucleic acid strand linked to n chemical moieties, where n is 1 to 10, and the nucleic acid conjugate products comprise a nucleic acid molecule linked to n+1 chemical moieties.

9. A method according to claim 1 wherein the nucleic acid or nucleic acid conjugate reactants are removed by contacting the reaction members with the binding member and separating from the reaction products nucleic acid or nucleic acid conjugate reactants that bind to the binding member.

10. (canceled)

11. A method according to claim 1 wherein the binding member is immobilised on a solid support.

12. A method according to claim 1 wherein the capture group comprises biotin or a biotin derivative.

13. A method according to claim 12 wherein the biotin derivative is desthiobiotin or iminobiotin.

14. A method according to claim 12 wherein the binding member comprises a biotin-binding protein.

15. A method according to claim 14 wherein the biotin-binding protein is streptavidin or avidin.

16. A method according to claim 2 wherein the chemical moiety comprises a second reactive group that reacts with the first reactive group to form a covalent bond and the capture molecule comprises a third reactive group that reacts with the first reactive group to form a covalent bond.

17. (canceled)

18. A method according to claim 16 wherein: (a) the first reactive group is an amine and the second and third reactive groups are activated carboxylic acid-derivatives, or vice versa; (b) the first reactive group is an aldehyde and one of the second and third reactive groups is an alkoxyamine or 1, 2-mercaptoamine and the other is a primary amine; or (c) the first reactive group is a chloroacetyl group and one of the second and third reactive groups is a nucleophilic group, such as thiol or phosphorothioate, and the other is a primary amine.

19-21. (canceled)

22. A method according to claim 1 further comprising (a) reacting the purified population of nucleic acid conjugates with a population of additional chemical moieties reactive with the chemical moiety of the nucleic acid conjugate, to produce reaction products comprising nucleic acid conjugate products comprising the additional chemical moiety and nucleic acid conjugate reactants, (b) contacting the mixture with a capping molecule that comprises a capture group, wherein the capping group reacts with the chemical moiety of the nucleic acid conjugate reactants in the reaction products to form covalent bonds, thereby attaching the capture group to nucleic acid conjugate reactants, and, (c) removing the unreacted nucleic acid conjugate reactants from the reaction products using a binding member that binds the capture group, thereby producing a purified population of nucleic acid conjugate products comprising the additional chemical moiety.

23. A method according to claim 22 comprising repeated steps (a) to (c) one or more times to produce a purified population of nucleic acid conjugate products comprising two or more additional chemical moieties.

24. (canceled)

25. A method of making a nucleic acid chemical library comprising: (a) providing a population of nucleic acid strands, each strand comprising a first reactive group, (b) reacting the nucleic acid strands with a diverse population of chemical moieties capable of reacting with the first reactive group to produce reaction products comprising a diverse population of nucleic acid conjugates that comprise nucleic acid strands conjugated to chemical moieties, (c) contacting the reaction products with a capping molecule that comprises a capture group, wherein the capping molecule reacts with the first reactive group of unreacted nucleic acid strands in the mixture to form covalent bonds, thereby attaching the capture group to the unreacted nucleic acid strands, (d) removing the unreacted nucleic acid strands from the reaction products using a binding member that binds the capture group, thereby producing a purified library of nucleic acid conjugates.

26. A method according to claim 25 wherein each nucleic acid strand comprises a coding sequence that encodes the chemical moiety that is conjugated thereto.

27. A method according to claim 25 further comprising (a) providing a purified library of nucleic acid conjugates comprising nucleic acid strands and chemical moieties, (b) reacting the library with a diverse population of additional chemical moieties to produce reaction products comprising a diverse population of nucleic acid conjugate products, each nucleic acid conjugate product comprising a combination of a chemical moiety and an additional chemical moiety, (c) contacting the reaction products with a capping molecule that comprises a capture group, such that the capping molecule reacts with the unreacted nucleic acid conjugates to form covalent bonds, thereby attaching the capture group to unreacted nucleic acid conjugates in the mixture, (d) removing the unreacted nucleic acid conjugates using a binding member that binds the capture group, thereby producing a purified library of nucleic acid conjugates comprising different combinations of chemical moieties and additional chemical moieties.

28. A method according to claim 27 further comprising repeating steps (a) to (d) to produce purified libraries of nucleic acid conjugates comprising different combinations of chemical moieties and multiple additional chemical moieties.

29. A method of making a nucleic acid chemical library comprising providing a plurality of reaction pools, each comprising nucleic acid strands comprising a first reactive group, contacting each pool with a different chemical moiety capable of reacting with the first reactive group of the nucleic acid strands to form covalent bonds, reacting the nucleic acid strands in each pool with the chemical moieties to form reaction products comprising nucleic acid conjugates comprising the chemical moieties, and either; (i) (a) combining the reaction products in the pools to produce a library of nucleic acid conjugates comprising nucleic acid strands linked to different chemical moieties, (b) contacting the library with a capping molecule comprising a capture group, wherein the capping molecule reacts with the first reactive groups of unreacted nucleic acid strands to form covalent bonds, and (c) removing unreacted nucleic acid strands from the library using a binding member that binds to the capture moiety to produce a purified library of nucleic acid conjugates, said nucleic acid conjugates comprising nucleic acid strands linked to different chemical moieties; or (ii) (a) contacting the reaction products in each pool with a capping molecule comprising a capture group, wherein the capping molecule reacts with the first reactive groups of unreacted nucleic acid strands to form covalent bonds, (b) removing unreacted nucleic acid strands using a binding member that binds to the capture moiety to produce purified populations of nucleic acid conjugates in each pool, and (c) combining the purified populations to produce a purified library of nucleic acid conjugates, said nucleic acid conjugates comprising nucleic acid strands linked to different chemical moieties.

30. A method according to claim 28 comprising: e) splitting the purified library into a plurality of pools, f) contacting each pool with a different additional chemical moiety, the additional chemical moiety being capable of reacting with the chemical moieties of the nucleic acid conjugates in the pool to form covalent bonds, g) reacting the nucleic acid conjugates in each pool with the additional chemical moiety to form reaction products comprising nucleic acid conjugate products, the nucleic acid conjugate products comprising combinations of a chemical moiety and an additional chemical moiety, each combination being conjugated to a nucleic acid strand, and either; (i) (h) combining the reaction products in the pools to produce a library of nucleic acid conjugates comprising combinations of chemical moieties and additional chemical moieties, each combination being conjugated to a nucleic acid strand, (i) contacting the library with a capping molecule comprising a capture group, wherein the capping molecule reacts with unreacted nucleic acid conjugate reactants to form covalent bonds, and (j) removing unreacted nucleic acid conjugate reactants from the library using a binding member that binds to the capture moiety to produce a purified library of nucleic acid conjugate products comprising a diverse population of combinations of chemical moieties and additional chemical moieties, each combination being conjugated to a nucleic acid strand; or (ii) (h) contacting the reaction products in each pool with a capping molecule comprising a capture group, wherein the capping molecule reacts with unreacted nucleic acid conjugate reactants to form covalent bonds, and (i) removing unreacted nucleic acid conjugate reactants from each pool using a binding member that binds to the capture moiety to produce a population of purified nucleic acid conjugate products in each pool, and (j) combining the populations of purified nucleic acid conjugate products in the pools to produce a library of nucleic acid conjugate products comprising combinations of chemical moieties and additional chemical moieties, each combination being conjugated to a nucleic acid strand.

31. A method according to claim 29 comprising repeating steps (e) to (j) to add one or more additional chemical moieties to produce a purified library comprising a diverse population of combinations of chemical moieties and two or more additional chemical moieties, each combination being conjugated to a nucleic acid strand.

32-34. (canceled)

Description

[0157] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

[0158] FIG. 1 shows a general scheme for the removal of nucleic acid (DNA) or nucleic acid conjugate reactants from nucleic acid conjugate products. In FIG. 1 X and Y indicate reactive groups. Z is a linker formed by the reaction of X and Y. R is a building block. ACG is an affinity capture group (also called a capture group) which is present on a capping molecule.

[0159] In FIG. 1, a modified DNA comprising a reactive group (Y), is reacted with the reactive group (X) of building block R. A successful reaction produces the desired DNA conjugate product. However, the reaction often does not go to completion leaving unreacted DNA strands as a major impurity in the reaction products. In order to remove the unreacted DNA, the reaction products are contacted with a capping molecule comprising a capture group (x-ACG). The capping molecule reacts with the reactive group present on the unreacted DNA to link the capture group to the unreacted DNA. The unreacted DNA can then be removed by a binding protein capable of binding to the capture group, while any unreacted building blocks can be removed by a washing step or ethanol precipitation of the DNA conjugates.

[0160] FIG. 2 shows a reaction scheme in which the capping molecule is an activated carboxylic-acid derivative of biotin. In FIG. 2 the DNA reactant is an amino modified DNA and the chemical moiety is a reactive (activated) small molecule carboxylic acid derivative. A successful acylation reaction between the amino modified DNA and an amino-reactive group (X) on the chemical moiety results in the formation of a peptide bond producing the desired DNA conjugate as a reaction product. Unreacted amino modified DNA is a major impurity in the reaction products. The unreacted amino modified DNA is contacted with a capping molecule comprising an activated carboxylic-acid derivative of biotin (for example an NHS-ester or mixed anhydride including Compound A or compound B)). The capping molecule reacts with the unreacted DNA to form a peptide bond. The unreacted DNA can then be removed by affinity capture using a biotin-binding protein (such as streptavidin) coated on a solid support, leaving a purified reaction mixture of the desired DNA conjugate.

[0161] FIGS. 3a and 3b show reactions which are useful for DNA-encoded chemical library synthesis.

[0162] FIG. 3a shows a reaction scheme in which the capping molecule is an aldehyde-reactive biotin derivative. The DNA reactant is an aldehyde-modified DNA and the chemical moiety comprises an amine. A reductive amination reaction between aldehyde-modified DNA and the amine group on the chemical moiety results in the formation of a hydrogen bond producing the desired DNA conjugate product. Unreacted aldehyde-modified DNA is a major impurity in the reaction products. Unreacted aldehyde-DNAs can be removed by the reaction with aldehyde-reactive biotin derivatives (e.g. alkoxyamines, 1, 2-mercaptoamines) followed by affinity selection with streptavidin on a solid support, for example streptavidin resin.

[0163] FIG. 3b shows a reaction scheme in which the capping molecule is a nucleophile containing biotin derivative. The DNA reactant is a chloroacetyl containing DNA and the chemical moiety comprises an amine. A nucleophilic displacement reaction between the chloroacetyl containing DNA and the amine group on the chemical moiety results in the formation of the desired DNA conjugate reaction product. Unreacted chloroacetyl-DNAs reaction products can be removed by the addition of a nucleophile containing biotin derivative (e. g. thiol, phosphorothioate) followed by capture on a solid support, for example streptavidin resin.

[0164] FIG. 4a shows the results of example 1 which was carried out according to method 1. Chemical moiety A352 (structure shown in FIG. 4a) was reacted with amino modified DNA. The bottom chromatogram shows that before purification, both unreacted DNA (DNA-NH2) and reaction product (DNA_NH-A352) are present in the reaction mixture. The middle chromatogram was carried out after contacting the reaction mixture with activated biotin (capping molecule). The top chromatogram was carried out after affinity capture on streptavidin to remove capped unreacted DNA-NH2. The % purity of the reaction product is as indicated.

[0165] FIG. 4b shows the results of example 2 which was carried out according to method 1. Chemical moiety A540 (structure shown in FIG. 4b) was reacted with amino modified DNA. The bottom chromatogram shows that before purification, both unreacted DNA (DNA-NH2) and reaction product (DNA_NH-A540) are present in the reaction mixture. The middle chromatogram was carried out after contacting the reaction mixture with activated biotin (capping molecule). The top chromatogram was carried out after affinity capture on streptavidin to remove capped unreacted DNA-NH2. The % purity of the reaction product is as indicated.

[0166] FIG. 5a shows the results of example 3 which was carried out according to method 1. Chemical moiety A548 (structure shown in FIG. 5a) was reacted with amino modified DNA. The bottom chromatogram shows that before purification, both unreacted DNA (DNA-NH2) and reaction product (DNA_NH-A548) are present in the reaction mixture. The middle chromatogram was carried out after contacting the reaction mixture with activated biotin (capping molecule). The top chromatogram was carried out after affinity capture on streptavidin to remove capped unreacted DNA-NH2. The % purity of the reaction product is as indicated.

[0167] FIG. 5b shows the results of example 4 which was carried out according to method 1. Chemical moiety A625 (structure shown in FIG. 5b) was reacted with amino modified DNA. The bottom chromatogram shows that before purification, both unreacted DNA (DNA-NH2) and reaction product (DNA_NH-A625) are present in the reaction mixture. The middle chromatogram was carried out after contacting the reaction mixture with activated biotin (capping molecule). The top chromatogram was carried out after affinity capture on streptavidin to remove capped unreacted DNA-NH2. The % purity of the reaction product is as indicated.

[0168] FIG. 6 shows the results of example 5 which was carried out according to method 2 and using chemical moiety number 1 from Table 1. FIG. 6 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number 1 from Table 1. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound A); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0169] FIG. 7 shows the results of example 6 which was carried out according to method 2 and using chemical moiety number 2 from Table 1. FIG. 7 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number 2 from Table 1. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound A); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0170] FIG. 8 shows the results of example 7 which was carried out according to method 2 and using chemical moiety number 3 from Table 1. FIG. 8 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number 3 from Table 1. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound A); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0171] FIG. 9 shows the results of example 8 which was carried out according to method 2 and using chemical moiety number 4 from Table 1. FIG. 9 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number 4 from Table 1. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound A); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0172] FIG. 10 shows the results of example 9 which was carried out according to method 2. The chemical moiety was number 5 from Table 1. FIG. 10 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number 5 from Table 1. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound A); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0173] FIG. 11 shows the results of example 10, which was carried out according to method 3 and using chemical moiety A335 (structure shown in figure). FIG. 11 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number A335. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound B); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs.

[0174] FIG. 12 shows the results of example 11, which was carried out according to method 3 and using chemical moiety A381 (structure shown in figure). FIG. 12 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number A381. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound B); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs.

[0175] FIG. 13 shows the results of example 12, which was carried out according to method 3 and using chemical moiety A490 (structure shown in figure). FIG. 13 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number A490. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound B); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs.

[0176] FIG. 14 shows the results of example 13, which was carried out according to method 3 and using chemical moiety A436 (structure shown in figure). FIG. 14 (top) shows a HPLC chromatogram of the reaction mixture following a reaction between DNA-NH2 and chemical moiety number A436. A peak is shown for the desired reaction product (prod) and the unreacted DNA reactant; (middle) HPLC chromatogram after capping of unreacted DNAs with a capping molecule (Compound B); (bottom) HPLC chromatogram after affinity capture to remove capped unreacted DNAs.

EXPERIMENTS

Materials and Methods

[0177] The desired DNA conjugates may be generated by the condensation of an amino modified DNA with an activated carboxylic acid to form an amide bond. As a general method, DEAE sepharose (Whatman, GE Healthcare) is washed with 10 mM aq. AcOH (2) and water (2). DNA-C.sub.12NH.sub.2 (1 nmol) is immobilized on the anion exchange resin by incubation of an aqueous solution of the DNA for 5 min, followed by washing with 10 mM eq. AcOH (2) and DMSO (2). A solution of an acid (50 mM), EDC (50 mM) and HOAt (5 mM) in DMSO (500 L) is preincubated for 5 min and added to the immobilized DNA. The reaction mixture is gently shaken for 2 h.

[0178] The resin is rinsed with DMSO (2). The coupling and washing steps are repeated (2). Then, the resin is washed with DMSO (2).

[0179] Capped nucleic acid conjugates were generated according to the following exemplary methods:

[0180] Method 1

[0181] A solution of biotin (free carboxylic acid) (50 mM), 1-Ethyl-3-(3-dimetylaminopropyl) carbodiimide (EDC, 50 mM) and 1-Hydroxy-7-azatriazole (HOAt, 5 mM) in DMSO (500 uL) is preincubated for 5 min and added to the immobilized DNA. The reaction mixture is shaken overnight (16 h). The resin is washed with DMSO (2) and 10 mM aq. AcOH (2). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.

[0182] Method 1 was used in examples 1-4 (FIGS. 4a-5b)

[0183] Method 2

[0184] Preparation of the Capping Molecule Compound A (Biotin Isobutyloxy Anhydride) and Capping of Unreacted Amine-DNA Conjugates

[0185] Diisopropylethylamine (DIPEA, 1.5 mmol) is added to a solution of carboxylic acid biotin (1 mmol) in dry DMSO (8 ml) under argon and stirred at 0 C. followed by the dropwise addition of isobutyl chloroformate (0.8 mmol). The reaction mixture is stirred for 1 h forming Compound A (biotin isobutyloxy anhydride) Freshly prepared Compound A is immediately added to the immobilized DNA (see above) and shaken overnight (16 h).

[0186] The resin is washed with DMSO (2) and 10 mM aq. AcOH (2). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.

[0187] Method 2 was used in examples 5-9 (FIGS. 6-10).

[0188] Method 3

[0189] A solution of biotin N-hydroxysuccinimidyl ester (compound B; 200 mM) and 4-dimethylaminoaniline (20 mM) in DMSO (200 uL) was added to the immobilized DNA (1-2 nmol). The reaction mixture is shaken for 4 h at 37 C. The resin is washed with DMSO (2) and 10 mM aq. AcOH (2). The DNA is eluted with AcOH buffer (3.0 M, pH 4.75) and precipitated with EtOH. Affinity capture of capped DNAs is performed as described below.

[0190] Method 3 was used in examples 10-13.

[0191] Affinity Capture:

[0192] Streptavidin Sepharose High Performance (Amersham Biosciences) is washed with H.sub.2O (2) and PBS (2). DNA solution (1.8 nmol) in PBS is added on the streptavidin resin and incubated for 15 min, then centrifuged (2). DNA conjugates are analysed directly by HPLC or further processed by EtOH precipitation.

[0193] Table 1 shows the chemical structures of chemical moieties 1-5 used in examples 5-9 (FIGS. 6-10). The purity of the reaction product before and after purification is also indicated.

TABLE-US-00001 TABLE 1 Affinity capture efficiencies (Method 2): Product Product fraction fraction after Before affinity affinity Chemical structures capture capture 1 [00002] 77% 100% 2 [00003] 45% 80% 3 [00004] 81% 95% 4 [00005] 79% 90% 5 [00006] 90% 100%

Examples

[0194] 1. Example 1 was carried out according to method 1. Chemical moiety A352 (structure shown in FIG. 4a) was reacted with amino modified DNA. Unreacted amino-modified DNA reactants were removed using biotin as a capping molecule followed by affinity capture on streptavidin. The results, including the % purity of the reaction product are shown in FIG. 4a.

[0195] 2. Example 2 was carried out according to method 1. Chemical moiety A540 (structure shown in FIG. 4b) was reacted with amino modified DNA. Unreacted amino-modified DNA reactants were removed using biotin as a capping molecule followed by affinity capture on streptavidin. The results, including the % purity of the reaction product are shown in FIG. 4b.

[0196] 3. Example 3 was carried out according to method 1. Chemical moiety A548 (structure shown in FIG. 5a) was reacted with amino modified DNA. Unreacted amino-modified DNA reactants were removed using biotin as a capping molecule followed by affinity capture on streptavidin. The results, including the % purity of the reaction product are shown in FIG. 5a.

[0197] 4. Example 4 was carried out according to method 1. Chemical moiety A625 (structure shown in FIG. 5b) was reacted with amino modified DNA. Unreacted amino-modified DNA reactants were removed using biotin as a capping molecule followed by affinity capture on streptavidin. The results, including the % purity of the reaction product are shown in FIG. 5b.

[0198] Examples 5-9 were carried out according to method 2. In examples 5-9, respectively, chemical moieties 1-5 from Table 1 were reacted with amino modified DNA. Unreacted amino-modified DNA was capped with a capping molecule (Compound A) and removed by affinity capture. The purity of the reaction product before and after affinity capture is shown in Table 1.

[0199] Example 10 was carried out according to method 3. Chemical moiety A335 (structure shown in FIG. 11) was reacted with amino modified DNA. Unreacted amino-modified DNA reactants were removed using biotin as a capping molecule followed by affinity capture on streptavidin. The results of the reaction product and subsequent streptavidin purification are shown in FIG. 11.

[0200] Examples 11-13 were carried out according to method 3. In examples 11-13, respectively, chemical moieties shown in FIGS. 12-14 (A381, A490, and A436) were reacted with amino modified DNA. Unreacted amino-modified DNA was capped with a capping molecule (Compound B) and removed by streptavidin affinity capture. The results of the reaction product and subsequent streptavidin purification are shown in FIGS. 12-14.

REFERENCES

[0201] 1 Mannocci, L. et al. PNAS USA 105(46):17670-17675 [0202] 2 Brenner, S. and Lerner, R. A. PNAS USA 89 (1992), 5381-5383 [0203] 3 Nielsen, J., et al., J. Am. Chem. Soc. 115 (1993) [0204] 4 Needels et al., M. C., PNAS USA 90 (1993), 10700-10704 [0205] 5 Gartner, Z. J., et al., Science 305 (2004), 1601-1605 [0206] 6 Melkko, S., et al., Nat. Biotechnol. 22 568-574 (2004) [0207] 7 Sprinz, K. I., et al., Bioorg. Med. Chem. Lett. 15 (2005), pp. 3908-3911 [0208] 8 Leimbacher et al Chemistry. 2012 Jun. 18; 18(25):7729-37 [0209] 9 Clark et al Nat Chem Biol. 2009 September; 5(9):647-54 [0210] 10 Wuts, P. G. L and Greene, T. W. Greene's Protective Groups in Organic Synthesis, Fourth Edition (2006).