title: The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system.
abstract: Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC-isoG pair. The fidelity-per-round of amplification was found to be approximately 98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only approximately 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA. msismour@chem.ufl.edu
authors.forename: A Michael
authors.initials: AM
authors.lastname: Sismour
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D020029:Base Pairing; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D006147:Guanine / Q000737:chemistry; D009841:Oligonucleotides / Q000096:biosynthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry; D013942:Thymine Nucleotides / Q000737:chemistry*
chemical_list: C504715:2-thiothymidine triphosphate; D017931:DNA Primers; D009841:Oligonucleotides; D013942:Thymine Nucleotides; C093270:isocytosine; C060625:2-thiothymidine; D006147:Guanine; D003596:Cytosine; C008183:isoguanine; D004256:DNA Polymerase I; C024157:thymidine 5'-triphosphate; D013936:Thymidine