title: Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet.
abstract: The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.
authors: Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors: Denis A
authors: DA
authors: Malyshev
authors: Kirandeep
authors: K
authors: Dhami
authors: Henry T
authors: HT
authors: Quach
authors: Thomas
authors: T
authors: Lavergne
authors: Phillip
authors: P
authors: Ordoukhanian
authors: Ali
authors: A
authors: Torkamani
authors: Floyd E
authors: FE
authors: Romesberg
mesh_terms: D001483:Base Sequence; D019295:Computational Biology; D004261:DNA Replication / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005818:Genetic Engineering / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D009711:Nucleotides; D009841:Oligonucleotides