Method for the discovery of high-affinity, high specificity oligonucleotide and derivatized oligonucleotide sequences for target recognition

1. A method of obtaining combimers that bind to a target at a desired binding affinity, comprising the steps (a) through (d) in the following order:

(a) designing an oligonucleotide library comprising oligonucleotides or analogs thereof having known primary and secondary structure, each of which comprises a constant region and a variable region comprising N enumerated base positions in a combimer loop region of the oligonucleotide or analog thereof, wherein each enumerated base position comprises one of X selected nucleotide bases, such that the oligonucleotide library comprises up to X N different oligonucleotides or analogs thereof, and wherein the nucleotide sequence is systematically modified to create a family of sequence variants, and wherein the region of the nucleotide sequence which is varied is said to be enumerated;

(b) synthesizing at least some of the oligonucleotides or analogs thereof of the oligonucleotide library;

(c) classifying or quantifying the binding affinity of the combimer loop region of each oligonucleotide or analog thereof for the target; and

(d) identifying combimers with the desired binding affinity from the library of oligonucleotides or analogs thereof,

wherein the combimer of step (d) is a member of the oligonucleotide library of (a) which comprises the known primary and secondary structure of step (a) and wherein N enumerated base positions comprises 2-40 nucleotides, and wherein the oligonucleotide library of (a) is reusable with multiple targets.

2. The method of claim 1 , further comprising:

selecting oligonucleotides or analogs thereof having an optimal binding domain from the library based upon the known primary structure of the combimer loop region of the identified combimer with desired binding affinity.

3. The method of claim 1 , wherein the oligonucleotides are chemically synthesized.

4. The method of claim 1 , wherein the oligonucleotides are enzymatically synthesized.

5. The method of claim 1 , wherein the oligonucleotides comprise DNA.

6. The method of claim 1 , wherein the oligonucleotides comprise RNA.

7. The method of claim 1 , wherein the oligonucleotides comprise both DNA and RNA in the same structure.

8. The method of claim 1 , further comprising catenating one or more combimers to produce a species with the desired binding affinity.

9. The method of claim 1 , wherein the combimer is chemically modified.

10. The method of claim 1 , wherein the enumerated region comprises 2-20 nucleotides.

11. The method of claim 10 , wherein the enumerated region comprises 2-10 nucleotides.

12. The method of claim 11 , wherein the enumerated region comprises 2-5 nucleotides.

13. The method of claim 1 , wherein the target is a protein.

14. The method of claim 1 , wherein the target is an organism.

15. The method of claim 14 , wherein the organism is a virus.

16. The method of claim 14 , wherein the organism is selected from the group consisting of cryptosporidium and giardia.

17. The method of claim 1 , wherein the target is a small molecule selected from the group consisting of toxins, environmental pollutants, drugs, and environmental contaminants.

18. The method of claim 1 , wherein binding affinity of the enumerated regions of the combimers is determined for more than one target.

19. The method of claim 1 , wherein the desired binding affinity is medium binding affinity of 10 −6 M<Kd<10 −2 M.

20. The method of claim 1 , wherein the desired binding affinity is high binding affinity of 10 −9 M<Kd<10 −6 M.

21. The method of claim 1 , wherein the loop is a stem loop or internal loop.

22. The method of claim 1 wherein the variable region is fully enumerated.

23. The method of claim 1 , further comprising repeating steps (b) through (d) with a larger enumerated region, N.

24. The method of claim 23 , wherein N is increased by 1-4 nucleotides.