Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2012; 109(30):12005-10. DOI: 10.1073/pnas.1205176109
Source: PubMed


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.

Download full-text


Available from: Phillip Ordoukhanian
    • "Fortunately, previous studies have demonstrated that codons containing unnatural base pairs are compatible with the E. coli translation system reconstituted in vitro [17] [78]. However, while thermostable PCR enzymes have been used for replication in vitro [67] [68] [69] [70] and E. coli PolI has been implemented in vivo [79], compatibility with E. coli PolIII replication has yet to be demonstrated. Additionally, T7 RNA polymerase is routinely used for transcription in vitro [74– 77], and E. coli RNA polymerase transcription has yet to be demonstrated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.
    No preview · Article · Sep 2015 · Journal of Molecular Biology
  • Source
    • "Recently, practical applications of orthogonal nucleic acids have been reported. XNA has been replicated in cell-free systems (e.g., PCR) (Yang et al., 2011; Betz et al., 2012; Malyshev et al., 2012), XNA has "
    [Show abstract] [Hide abstract]
    ABSTRACT: Synthetic Biology promises low-cost, exponentially scalable products and global health solutions in the form of self-replicating organisms, or "living devices." As these promises are realized, proof-of-concept systems will gradually migrate from tightly regulated laboratory or industrial environments into private spaces as, for instance, probiotic health products, food, and even do-it-yourself bioengineered systems. What additional steps, if any, should be taken before releasing engineered self-replicating organisms into a broader user space? In this review, we explain how studies of genetically modified organisms lay groundwork for the future landscape of biosafety. Early in the design process, biological engineers are anticipating potential hazards and developing innovative tools to mitigate risk. Here, we survey lessons learned, ongoing efforts to engineer intrinsic biocontainment, and how different stakeholders in synthetic biology can act to accomplish best practices for biosafety.
    Full-text · Article · Jan 2013 · Frontiers in Microbiology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA has been around for billions of years — but that doesn't mean scientists can't make it better.
    Preview · Article · Nov 2012 · Nature
Show more