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.expanded genetic alphabet | hydrophobic | artificial DNA | unnatural nucleotides | bioinformatics E xpansion of the genetic alphabet to include an unnatural base pair has emerged as a central goal of chemical and synthetic biology. Success would represent a remarkable integration of orthogonal synthetic components into a fundamental biological system and build the foundation for a semisynthetic organism with increased potential for information storage and retrieval (1). Moreover, the constituent unnatural nucleotides could be used to site-specifically label DNA or RNA with different functionalities of interest (2-4) and potentially revolutionize the already ubiquitous in vitro applications of nucleic acids, such as aptamer and DNA/RNAzyme selections (5, 6), PCR-based diagnostics (7, 8), and DNA-based nanomaterials and devices (9).Although many candidate unnatural base pairs have been reported (10-21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context. Sequence dependencies would cause biased amplification and effectively preclude many uses of the unnatural base pair. No candidate unnatural base pair has been shown to be replicated without sequence bias, and thus, none can yet claim functional equivalence to a natural base pair.In general, the most promising unnatural base pair candidates currently available have been developed by pursuing one of two different strategies. The first strategy, pioneered in the work by Benner and coworkers (22), relies on the use of nucleotide analogs bearing nucleobases that pair through complementary hydrogen bonding (H-bonding) patterns that are orthogonal to those patterns of the natural pairs. Early ef...

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

“…1A) being a particularly promising example (5)(6)(7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7)(8)(9)(10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first progress toward the creation of an SSO in 2014, when we reported that Escherichia coli grown in the presence of the corresponding unnatural nucleoside triphosphates (dNaMTP and d5SICSTP), and provided with a plasmid-encoded nucleoside triphosphate transporter (NTT2) from Phaeodactylum tricornutum (which we denote as PtNTT2) (13), is able to import the unnatural triphosphates and replicate a single dNaM-d5SICS UBP on a second plasmid (14).…”

Library construction for next-generation sequencing: Overviews and challenges

“…This form of targeted sequencing is more appropriate for applications such as microbiomic experiments where community composition is analyzed by surveying 16S rRNA sequences in complex bacterial mixtures (57), analysis of antibody diversity (58) and T cell receptor gene repertoires (50), and facilitating the process of identifying and selecting high value aptamers in a SELEX protocol (59). To highlight the flexibility of amplicon sequencing, a recent study used the method to analyze the incorporation of unnatural nucleotides during DNA synthesis (60). …”

Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology

“…In fact, d NaM -d 5SICS was the first of our UBP candidates to be amplified in a standard PCR reaction with high efficiency and fidelity. 32 To determine whether d NaM is optimal for pairing with d 5SICS , we further explored derivatization of the d MMO2 scaffold with Cl, Br, I, Me, and Pr meta-substituents, but none improved replication. 33 We next examined 28 analogs with different para-substituents, and the SAR was augmented with PCR amplification assays using Taq, OneTaq, and KOD polymerases (Figure 9A).…”