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(12) United States Patent (10) Patent No.: US 7,794,936 B1 Benner Et Al

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(12) United States Patent (10) Patent No.: US 7,794,936 B1 Benner Et Al US007794936B1 (12) United States Patent (10) Patent No.: US 7,794,936 B1 Benner et al. (45) Date of Patent: Sep. 14, 2010 (54) USING THYMIDINE ANALOGS TO IMPROVE Michiko Kimoto, Rie Kawai, Tsuneo Mitsui, Shigeyuki Yokoyama, REPLICATION IN AN EXPANDED GENETIC Ichiro Hirao An unnatural base pair system for efficient PCR ampli ALPHABET fication and functionalization of DNA molecules Nucleic Acids Research, 2009, vol. 37, No. 2 e14. (76) Inventors: Steven Albert Benner, 1501 NW. 68" Chen, F., Gaucher, E. A., Leal, N. A., Hutter, D., Havemann, S.A., Govindarajan, S., Ortlund, E. A., Benner, S.A. (2010) Reconstructed Ter, Gainesville, FL (US) 32605-4147: evolutionary adaptive paths give polymerases accepting reversible Albert Michael Sismour, 85 Hancock terminators for sequencing and SNP detection. Proc. Natl. Acad. Sci. St., Apt. 7, Cambridge, MA (US) 02139 USA 107, 5, 1948-1953. (*) Notice: Subject to any disclaimer, the term of this * cited by examiner patent is extended or adjusted under 35 U.S.C. 154(b) by 534 days. Primary Examiner Heather Calamita (21) Appl. No.: 11/541.295 (57) ABSTRACT (22) Filed: Sep. 29, 2006 This invention relates to nucleoside, nucleotide, and oligo (51) Int. Cl. nucleotide analogs that incorporate non-standard nucleobase CI2O I/68 (2006.01) analogs, those that present a pattern of hydrogen bonds to a paired nucleobase analog in a complementary strand that is (52) U.S. Cl. ........................ 435/6:536/22.1536/24.33 different from the pattern presented by adenine, guanine, (58) Field of Classification Search ....................... None cytosine, and thymine. Most specifically, this invention dis See application file for complete search history. closes and claims processes for amplifying nucleic acid ana (56) References Cited logs containing non-standard nucleobases using polymerase chain reactions, and combinations of non-standard nucleo U.S. PATENT DOCUMENTS bases, analogs of standard nucleotides, and enzymes that 6,977,161 B2 * 12/2005 Grenier et al. ............. 435,912 perform this amplification. Most specifically, this invention is for the use of 2-thiothymidine triphosphate (2-thioTTP) OTHER PUBLICATIONS instead of thymidine triphosphate in a six letter polymerase Sismour et al. Nucleic Acids Research vol. 32:728-735. 2004. chain reaction that includes 2'-deoxyadenosine triphosphate, Horlacher, J., Hottiger, M., Podust, V. N., Huebscher, U. Benner, S. 2'-deoxyguanosine triphosphate, 2'-deoxycytidine triphos A. (1995) Recognition by viral and cellular DNA polymerases of phate, 2'-deoxy-iso-guanosine triphosphate, and 2'-deoxy nucleosides bearing bases with non-standard hydrogen bonding pat iso-cytidine triphosphate, as well as their forms that contain terns. Proc. Natl. Acad. Sci. USA 92,6329-6333. side chain modifications. Because of the size and hydrogen Sismour, A. M., Lutz, S. Park, J.-H. Lutz, M. J., Boyer, P. L., bonding properties of the sulfur unit in 2-thioT, 2-thioT does Hughes, S. H. Benner, S. A. (2004) PCR amplification of DNA containing non-standard base pairs by variants of reverse not mispair effectively with the minor tautomer of isoG. This transcriptase from human immunodeficiency virus-1. Nucleic Acids. permits the PCR amplification of a six letter artificially Res.32, 2,728-735. expanded genetic information system, we examined the rela Sismour, A.M., Benner, S.A. (2005) The use ofthymidine analogs to tive rates of misincorporation of 2-thioTTP and TTP opposite improve the replication of an extra DNA base pair. A synthetic bio isoG using affinity electrophoresis with a fidelity-per-round logical system. Nucleic Acids Res. 33, 17. 5640-5646. of ca. 98%. The analogous PCR employing TTP has a fidel Yang, Z., Hutter, D., Sheng, P., Sismour, A. M., Benner, S.A. (2006) ity-per-round of only ca. 93%. Therefore, this invention rep Artificially expanded genetic information system: A new base pair resents the first example of a six letter artificial genetic system with an alternative hydrogen bonding pattern. Nucleic Acids Res. 34. that is amplifiable by a thermostable polymerase, and capable 21, 6095-6101. of Darwinian evolution. Yang, Z., Sismour, A. M. Sheng, P. Puskar, N. L., Benner, S. A. (2007) Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 35, 13,4238-4249. 8 Claims, 6 Drawing Sheets U.S. Patent Sep. 14, 2010 Sheet 1 of 6 US 7,794,936 B1 Figure 1. U.S. Patent Sep. 14, 2010 Sheet 2 of 6 US 7,794,936 B1 Figure 2. isoG minor tautomer 2-thioT Steric Clash Steric Clash U.S. Patent Sep. 14, 2010 Sheet 3 of 6 US 7,794.936 B1 Figure 3. P 0.5 o 3.6 0.5 1.0 3.0 os to 3.0 0.5 1.0 3.0 Time - - GTP, i-CTP GTP TTP GTP, 2-thioTTP GTP (min) U.S. Patent Sep. 14, 2010 Sheet 4 of 6 US 7,794,936 B1 Figure 4. template base A isoG P- S P+ TiC k is primer (P) t- as f is . sue dNTP T S TS iC,S TS U.S. Patent Sep. 14, 2010 Sheet 5 of 6 US 7,794,936 B1 Figure 5. 200 bp 100 bp 50 bp STP TTP TTP MM U.S. Patent Sep. 14, 2010 Sheet 6 of 6 US 7,794,936 B1 Figure 6. PCR Fidelity 1 OO 8 40 2 -3-2-thioP -- TTP 5 O 15 2 25 RCS Of PCR US 7,794,936 B1 1. 2 USING THYMIDINEANALOGSTOIMPROVE standard A, G, T, C, but little else with efficiency and high REPLICATION IN AN EXPANDED GENETIC fidelity Goo93 MorOOTab95 Mey04. ALPHABET Sismour et al. Sis04 recently reported the PCR amplifi cation of DNA containing a pair between 2,4-diaminopyri STATEMENT OF RIGHTS TO INVENTIONS 5 midine and Xanthine (called the pyl)AD:puADA base pair, MADE UNDER FEDERALLY SPONSORED because the pyrimidine implements the hydrogen bond RESEARCH Donor-Acceptor-Donor hydrogen bond pattern, from the major groove to the minor groove, complementary to the This invention was made with government Support under Acceptor-Donor-Acceptor pattern implemented by the CHE0434507 awarded by the National Science Foundation's 10 purine Xanthine). This was achieved using a double mutant of Center for Chemical Bonding. The government has certain the reverse transcriptase from human immunodeficiency rights in the invention virus (HIV) Ithat was obtained by a combination of inclinico selection and rational design. This mutant amplified an oli CROSS REFERENCE TO RELATED gonucleotide containing a single pyDAD over five rounds of APPLICATIONS 15 PCR with an overall fidelity (per round) of >99% for the pyDAD:puADA base pair. This process has only narrow util None ity, however, as the reverse transcriptase is not stable to heat ing, and therefore must be added anew after each heat cycle. NAMES OF THE PARTIES TO AJOINT A second PCR amplification of DNA containing an iso-C: RESEARCH AGREEMENT iso-G (py AAD:puldDA) nucleobase pair was achieved for several dozen rounds of amplification Jooh04 using a frag Not applicable. ment of DNA polymerase I from Thermus aquaticus that does not have an 5'-->3' exonuclease domain, with a fidelity of only INCORPORATION-BY-REFERENCE OF ~96% per round. The T. aquaticus polymerase is stable MATERIAL SUBMITTED ON ADISC 25 against thermal denaturation. A fidelity of less than 98% per round is not adequate for most practical applications, includ None. ing using the six letter alphabet as part of an in vitro selection system. FIELD Similar problems are encountered where steric comple 30 mentarity is used as the basis for nucleobase pairing specific This invention relates to the field of nucleic acid chemistry, ity Mor97Ber00 Hiro4). more specifically to the field of compositions and processes The loss of fidelity in the system reported by Joh04 is that can be used to amplify nucleic acid analogs. More spe most likely due to a mispairing of TTP opposite isoG. This cifically, this invention relates to compositions that allow the mispairing was found in earlier work by Switzer et al. 35 Swi93. It is also expected, given the long known fact that artificial selection based on artificial genetic systems. isoG has a minor tautomeric form that is complementary to T. BACKGROUND and present in aqueous solution to the extent of ca. 10%. BRIEF DESCRIPTION OF THE DRAWINGS Artificially expanded genetic information systems, often 40 known by the acronym AEGIS, have been widely sought FIG. 1. Artificially expanded genetic information system Sis04 Joh04Liu04 Hen04 Ben04 Ben05, including in used in this study. the patent literature (U.S. Pat. No. 5,432.272, for “Method for FIG. 2. Steric exclusion of 2-thioT-isoG base pair. Shown incorporating into a DNA or RNA oligonucleotide using is the mispair between thymidine (T) and the minor tautomer nucleotides bearing heterocyclic bases”; U.S. Pat. No. 6,001, 45 of isoG, resulting in replication infidelity. Shown as well is 983, for “Oligonucleotides with non-standard bases and the pair between 2-thiothymidine (2-thioT) and 2-aminoad methods for preparing same''); U.S. Pat. No. 6,037,120, for enine, showing a steric clash in the minor-groove. Similarly, "Recognition of oligonucleotides containing non-standard a steric clash between 2-thiol and the minor tautomer of isoG base pairs”; U.S. Pat. No. 6,140,496, for “Precursors for is expected to inhibit formation of this mispair. deoxyribonucleotides containing non-standard nucleosides: 50 FIG. 3. Primer extension reactions comparing 2-thioTTP and U.S. Pat. No. 5,965,364, for “Method for selecting func and TTP incorporation opposite isoG. Taq polymerase was tional deoxyribonucleotide derivatives'). They are also being incubated with template (T-1), primer (P-1), dGTP, and either mentioned in the popular press Bal04Gib04. A variety of isoCTP, TTP, or 2-thioTTP at 72°C.
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