Efforts toward Expansion of the Genetic Alphabet:  DNA Polymerase Recognition of a Highly Stable, Self-Pairing Hydrophobic Base

McMinn, Dustin L.; Ogawa, Anthony K.; Wu, Yun; Liu, Jianquan; Schultz, and Peter G.; Romesberg, Floyd E.

doi:10.1021/ja9925150

Cited by 258 publications

(213 citation statements)

References 28 publications

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“…These populations are consistent with the observed X-ray structure. In addition to rapid ring flipping, additional self pair motion is suggested by line broadening of intranucleotide NOE cross peaks of the flanking nucleobases dA 5 and dG 10 , and dA 5 imino resonance broadening which may result from enhanced solvent exchange, possibly due to increased solvent accessibility or base pair fluctuations. 38 Such fluctuations may be coupled to the observed ring flipping of the d3FB analogs.…”

Section: Resultsmentioning

confidence: 99%

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

et al. 2007

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Expansion of the genetic alphabet has been a long time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semi-synthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogs, such as propinyl isocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogs inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogs that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluoro benzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self pair formed between two d3FB analogs is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analog (dBEN). In all, the data suggest that structure, electrostatics and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.

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Section: Resultsmentioning

confidence: 99%

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

et al. 2007

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“…The amplification of DNA containing multiple, contiguous unnatural base pairs has also been shown (10), but the more general effects of sequence context have yet to be reported. The second strategy, originally pursued by our group (25), and in part inspired by the observation in the work by Kool and coworkers (26) that H-bonds are not absolutely required for polymerase recognition, relies on harnessing hydrophobic and packing forces between the nucleobase analogs. This approach was also pursued in the works by Hirao and coworkers (13,27), which developed a base pair that is amplified through PCR but with significant sequence bias.…”

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

Malyshev

Dhami²,

Quach³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…24,25 Recently, the Schultz and Romesberg laboratories have described a number of quite successful examples, including stably hybridizing self-pairs and cross-pairs, 55 and Ishikawa et al have also contributed new base pairs. 56 We have proposed that the most important criterion of a polymerase for a successful base pair is that it fit into the tight, rigid active site as would a normal pair.…”

Section: Figurementioning

confidence: 99%

Replacing the Nucleobases in DNA with Designer Molecules

Kool

2003

ChemInform

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Efforts toward Expansion of the Genetic Alphabet: DNA Polymerase Recognition of a Highly Stable, Self-Pairing Hydrophobic Base

Cited by 258 publications

References 28 publications

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

Efforts toward Expansion of the Genetic Alphabet: Structure and Replication of Unnatural Base Pairs

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

Replacing the Nucleobases in DNA with Designer Molecules

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