Binding and Catalytic Molecules Built from L-DNA with Added Nucleotides

Abstract

This invention provides for processes for binding to and/or chemically transforming a preselected target, where the process involves contacting said target to an oligonucleotide molecule that contains one or more “non-standard” nucleotides, which are nucleotide analogs that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. This disclosure provides an example where such an oligonucleotide molecule is built from both D- and L-mirror image carbohydrates in the backbone. It also provides a process for obtaining these binders and/or transformers by a laboratory in vitro selection process that exploits rolling circle amplification rather than the polymerase chain reaction.

Claims

1. A process for binding to a preselected target, said process comprising contacting said target with an oligonucleotide that comprises one or more nucleotides, wherein at least one nucleobase of said nucleotide has a nucleobase selected from the group consisting of ##STR00001## wherein R is the point of attachment of said nucleobase to said oligonucleotide, wherein said target is not Watson-Crick complementary to said oligonucleotide, and wherein the carbohydrates of said oligonucleotide have either D-configurations or L-configurations.

2. The process of claim 1, wherein said nucleobase is ##STR00002## where R is the point of attachment of said nucleobase to said oligonucleotide.

3. The process of claim 1, wherein said nucleobase is ##STR00003## where R is the point of attachment of said nucleobase to said oligonucleotide.

4. The process of claim 1, wherein said nucleobase is ##STR00004## where R is the point of attachment of said nucleobase to said oligonucleotide.

5. The process of claim 1, wherein said nucleobase is ##STR00005## where R is the point of attachment of said nucleobase to said oligonucleotide.

6. The process of claim 1, wherein said nucleobase is ##STR00006## where R is the point of attachment of said nucleobase to said oligonucleotide.

7. The process of claim 1, wherein said nucleobase is ##STR00007## where R is the point of attachment of said nucleobase to said oligonucleotide.

8. The process of claim 1, wherein said nucleobase is ##STR00008## where R is the point of attachment of said nucleobase to said oligonucleotide.

9. The process of claim 1, wherein said nucleobase is ##STR00009## where R is the point of attachment of said nucleobase to said oligonucleotide.

10. The process of claim 1, wherein said nucleobase is ##STR00010## where R is the point of attachment of said nucleobase to said oligonucleotide.

11. The process of claim 1, wherein said nucleobase is ##STR00011## where R is the point of attachment of said nucleobase to said oligonucleotide.

12. The process of claim 1, wherein said nucleobase is ##STR00012## where R is the point of attachment of said nucleobase to said oligonucleotide.

13. The process of claim 1, wherein said nucleobase is ##STR00013## where R is the point of attachment of said nucleobase to said oligonucleotide.

14. The process of claim 1, wherein said nucleobase is ##STR00014## where R is the point of attachment of said nucleobase to said oligonucleotide.

15. A process for chemically transforming a preselected target, said process comprising contacting said target with an oligonucleotide that comprises one or more nucleotides, wherein at least one nucleobase of said nucleotide has a nucleobase selected from the group consisting of ##STR00015## wherein R is the point of attachment of said nucleobase to said oligonucleotide, wherein said target is not Watson-Crick complementary to said oligonucleotide, and wherein the carbohydrates of said oligonucleotide have either D-configurations or L-configurations.

16. The process of claim 15, wherein said nucleobase is ##STR00016## where R is the point of attachment of said nucleobase to said oligonucleotide.

17. The process of claim 15, wherein said nucleobase is ##STR00017## where R is the point of attachment of said nucleobase to said oligonucleotide.

18. The process of claim 15, wherein said nucleobase is ##STR00018## where R is the point of attachment of said nucleobase to said oligonucleotide.

19. The process of claim 15, wherein said nucleobase is ##STR00019## where R is the point of attachment of said nucleobase to said oligonucleotide.

20. The process of claim 15, wherein said nucleobase is ##STR00020## where R is the point of attachment of said nucleobase to said oligonucleotide.

21. The process of claim 15, wherein said nucleobase is ##STR00021## where R is the point of attachment of said nucleobase to said oligonucleotide.

22. The process of claim 15, wherein said nucleobase is ##STR00022## where R is the point of attachment of said nucleobase to said oligonucleotide.

23. The process of claim 15, wherein said nucleobase is ##STR00023## where R is the point of attachment of said nucleobase to said oligonucleotide.

24. The process of claim 15, wherein said nucleobase is ##STR00024## where R is the point of attachment of said nucleobase to said oligonucleotide.

25. The process of claim 15, wherein said nucleobase is ##STR00025## where R is the point of attachment of said nucleobase to said oligonucleotide.

26. The process of claim 15, wherein said nucleobase is ##STR00026## where R is the point of attachment of said nucleobase to said oligonucleotide.

27. The process of claim 15, wherein said nucleobase is ##STR00027## where R is the point of attachment of said nucleobase to said oligonucleotide.

28. The process of claim 15, wherein said nucleobase is ##STR00028## where R is the point of attachment of said nucleobase to said oligonucleotide.

29. A process for extracting from a mixture of oligonucleotide molecules, whose members have unknown sequences, specific oligonucleotide molecules that bind to a preselected target, wherein said specific oligonucleotide molecules comprise one or more non-standard nucleotides wherein the nucleobase(s) of said nucleotide(s) has or have a nucleobase selected from the group consisting of ##STR00029## wherein R is the point of attachment of said nucleobase to said oligonucleotide, wherein said target is not Watson-Crick complementary to said specific oligonucleotide molecules, wherein said process comprises (a) obtaining said mixture of oligonucleotides having preselected regions containing, at positions that are not preselected, one or more of said non-standard nucleotides, (b) contacting said mixture with said target, (c) separating the oligonucleotides in said mixture having a enhanced affinity to the target molecule relative to the mixture from the remainder of the oligonucleotides in the mixture; and (d) amplifying the separated oligonucleotides using rolling circle amplification.

30. A process for extracting from a mixture of oligonucleotide molecules, whose members have unknown sequences, specific oligonucleotide molecules that chemically transform to a preselected target, wherein said specific oligonucleotide molecules comprise one or more non-standard nucleotides wherein the nucleobase(s) of said nucleotide(s) has or have a nucleobase selected from the group consisting of ##STR00030## wherein R is the point of attachment of said nucleobase to said oligonucleotide, wherein said target is not Watson-Crick complementary to said specific oligonucleotide molecules, wherein said process comprises (a) obtaining a mixture of oligonucleotides having preselected regions containing, at positions that are not preselected, one or more of said non-standard nucleotide analogs, (b) contacting said mixture with said target, (c) separating the oligonucleotides in said mixture having a greater effectiveness to transform said target reaction relative to the mixture from the remainder of the oligonucleotides in the candidate mixture; and (d) amplifying the separated oligonucleotides using rolling circle amplification.

Description

(h) BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1. Watson-Crick pairing rules follow two rules of complementarity: (a) size complementarity (large purines pair with small pyrimidines) and (b) hydrogen bonding complementarity (hydrogen bond donors, D, pair with hydrogen bond acceptors A). Rearranging D and A groups on various heterocycles supports an artificially expanded genetic information system (AEGIS). AEGIS nucleobases can also be functionalized at the position indicated by the “R” in these structures. These are the presently most preferred heterocycles for the instant invention. The nucleotides herein that are not found in natural DNA or RNA are called “non-standard”.

[0028] FIG. 2. This invention teaches the different heterocycles can implement the same hydrogen bonding pattern. Shown here are presently preferred heterocycles for implementing the instant invention, as alternatives. The nucleotides herein that are not found in natural DNA or RNA are called “non-standard”.

[0029] FIG. 3. Synthetic route generates a glycal having the L-configuration. This is a precursor for synthesizing all C-glycosides in the instant invention, specifically from FIG. 1, the heterocycles implementing the V, K. and Z hydrogen bonding patterns, and in FIG. 2, the heterocycles implementing the S, V, K, and Z hydrogen bonding patterns following routes covered in the parents of the instant application. This drawing also described the route to prepare the FIG. 1 AEGIS S in its epimeric form.

[0030] FIG. 4. Synthetic route to a protected phosphoramidite of AEGIS Z follows routes that are described in the parents. The precursor is commercially available from Carbosynth.

[0031] FIG. 5. Synthetic route to generate L-AEGIS P. Other AEGIS N-glycoside analogs are prepared analogously following routes described for the D-enantiomer in the parents.

[0032] FIG. 6. Rolling circle amplification (RCA) is disclosing this diagram as an alternative to PCR way to amplify survivors from an AEGIS-LIVE, the process disclosed in the parents. Here, instead of the primer binding sites (PBS) being used to bind primers in the PCR, those same sites are used to anneal to a splint which allows the ligation of the survivors to form a circle. The splint can then be used as a primer for rolling circle amplification, leading to between 10,000 and 100.000 copies of the survivor sequence(s) as a linear concatamer. These can then be cleaved by a restriction enzyme placed strategically to yield the same number of short survivor sequence(s) which can be introduced into AEGIS-LIVE. The presently preferred polymerase for the RCA is Bst I or other polymerase disclosed in U.S. Pat. No. 90,602,345, also a descendent of U.S. patent application Ser. No. 12/999,138, which is incorporated herein by reference is entirely.

DESCRIPTION OF INVENTION

Definition of Non-Standard Components of an Artificially Expanded Genetic Information System

[0033] This application teaches a distinction between the hydrogen-bonding pattern (in FIG. 1 and FIG. 2 nomenclature, pyDAD, for example) and the heterocycle that implements it. Thus, the pyADA hydrogen-bonding pattern is implemented by thymidine, uridine, and pseudouridine. The puDDA hydrogen bonding pattern is implemented by both the heterocycle isoguanosine and 7-dear-isoguanosine. Heterocycles to implement any particular pre-selected hydrogen-bonding pattern are preferred depending on their chemical properties, for example, high chemical stability or low tautomeric ambiguity. The pyADA, pyDAA, puADD, and puDAD hydrogen bonding patterns are said to be “standard” hydrogen bonding patterns, and to form with their appropriate complement “standard base pairs”. Other hydrogen bonding patterns are said to be “non-standard”, and to form with their appropriate complement “non-standard base pairs”.

Creating AEGIS-Containing Oligonucleotides Having all of the Carbohydrate Suitors in the L-Configuration

[0034] The strategy to generate binding and reactive AEGIS sequences that are stable in cancer-relevant biological environments relies in our ability to present them in their mirror-image form. These in AEGIS-free oligonucleotides are not be substrates for any natural nucleases, including those found in human blood and tissues; mirror image xNA is stable in blood, for example, for as long as 72 hours [Kim, K. R., Lee, T., Kim, B. S., & Ahn, D. R. (2014). Utilizing the bioorthogonal base-pairing system of L-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos. Chem. Sci. 5, 1533-1537.].

[0035] Synthesis of L-AEGIS oligonucleotides is implemented using solid phase phosphoramidite chemistry, well known in the art. The only difference is that the phosphoramidite building blocks have the L configuration, and are prepared by one of the processes described in the drawings.

[0036] The sequence for the L-AEGIS oligonucleotide(s) that bind and/or chemically transform to an achiral target is obtained simply by following the processes disclosed in the parents. Since the target is not chiral, the L-AEGIS oligonucleotide will bind to and/or chemically transform that achiral target with exactly the same affinity and/or exactly the same rate as the D-AEGIS oligonucleotide.

[0037] The sequence for the L-AEGIS oligonucleotide(s) that bind and/or chemically transform to a chiral target is obtained by following the processes disclosed in the parents, except by using the target in its mirror image enantiomeric form. The processes disclosed in the parents wilt generate D-AEGIS oligonucleotide(s) that bind to and/or chemically transform that chiral target in the form that is the mirror image of the desired target, Then, by symmetry laws in physics, the L-AEGIS oligonucleotide will bind to and/or chemically transform the chiral target in the desired enantiomeric form with exactly the same affinity and/or exactly the same rate as the D-AEGIS oligonucleotide binds to and/or transforms the target in its mirror image enantiomeric form.

[0038] When the desired target is a natural translated protein, which is built from L-amino acids, the target must be the same protein sequence, except built from D-amino acids, Kent and his colleagues have used convergent synthesis to make mirror-image proteins that are arbitrarily large [Kent, S. B. (2009). Total chemical synthesis of proteins. Chemical Society Reviews 38, 338-351.]. His technology is an alternative should we encounter the pitfall that no loops create AEGISbodies. Alternatively, AEGIS-LIVE may be targeted against a surface loop peptide of a target protein, preferably a flexible surface loop peptide, in the target protein, but synthetic so that it is built from D-amino acids [Rowlands, D. J., Clarke, B. E., Carroll, A. R., Brown, F., Nicholson, B. H., Bittle, J. L., Houghten, R. A. & Lerner, R. A. (1983) Nature (London) 306, 694-697.] [Alexander, H., Johnson, D. A., Rosen, J., Jerabek, L., Green, N., Weissman, I. L. & Lerner, R. A. (1983) Nature (London) 306, 697-699.] [Geysen, H. M., Barteling, S. J., & Meloen, R. H. (1985). Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc. Natl. Acad. Sci. USA 82, 178-182.]. This presently preferred process follows rules to how to extract peptides from a full protein to serve for this purpose [Walter, G. (1986). Production and use of antibodies against synthetic peptides, J. Immunol. Meth. 88, 149-161.

[0039] To force the peptide to adopt a turn conformation, the presently preferred implementation places cysteines at the end of the peptide. These form a cyclic disulfide is conformation resembles that of the natural were turned in a natural protein. Again, the amino acids must have the D-configuration.

[0040] The process whereby PCR is replaced by rolling circle amplification (RCA) is disclosed in FIG. 6. The presently preferred polymerase for the RCA is Bst I or other polymerase disclosed in U.S. Pat. No. 90,602,345, also a descendent of U.S. patent application Ser. No. 12/999,138, which is incorporated herein by reference is entirely. Not disclosed in U.S. Pat. No. 90,602,345, and disclose here for the first time, is the use of the sequences that would be, in PCR amplification, the primer binding sites, instead to template the ligation to form a circle with a splint complement. That may, optionally, also be used as the primer for the RCA. Finally, after the concatamer is created, the short survivors can be generated by digestion by restriction endonuclease at a restriction site placed strategically in the primer binding sites. The short survivors can then be put into the next cycle of AEGIS-LIVE.

[0041] Further loss of isoguanosine (B) species can be mitigated by using 7-deazaisoguanosine in the RCA process, or by using thiothyrnidine in the process to suppress mispairing between thymidine and isoguanosine and isoguanosine analogs in the template. Both of these are in the most preferred embodiments in the structure shown in FIG. 1.

EXAMPLES

Example 1

Synthesis of a Glycal Having the Unnatural L-Configuration

[0042] The mirror image L-nucleotides for G, A, T, and C needed to construct the L-AEGISbodies are commercially available. However, the L-AEGIS nucleotides that are used to synthesize the L-AEGISbodies are not. The precursor for the AEGIS components that are C-glycosides is prepared by the literature route shown in FIG. 3. Arabinose, a very inexpensive L-sugar, is the precursor. The same route, adapted from the parents for the L-glycal, leads to the formation of RNA AEGIS building blocks.

Example 2

Synthesis of a L-AEGIS Nucleoside Phosphoramidites Suitable for Solid Phase DNA Synthesis, here Implementing the Z Hydrogen Bonding Pattern from FIG. 2

[0043] An alternative route to C-glycosidic AEGIS components that are C-glycosides is shown in FIG. 4. This alternative route starts from L-thymidine, which is presently available from Carbosynth for $750/25 grams. This also follows synthetic procedures disclose in the parents. The inversion of configuration does not change any of the chemistry involved.

Example 3

Synthesis of a L-AEGIS Nucleoside Phosphoramidites Suitable for Solid Phase DNA Synthesis, here Implementing the P hydrogen Bonding Pattern from FIG. 1 and FIG. 2

[0044] The route to prepare N-glycosidic AEGIS components in a protected form. This starts with the-L chlorosugar. Again, this follows synthetic procedures disclose in the parents. The inversion of configuration does not change any of the chemistry involved.