Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs

Lu, Haige; Krueger, Andrew T.; Gao, Jianmin; Li, Haibo; Kool, Eric T.

doi:10.1039/c002766a

Cited by 25 publications

(37 citation statements)

References 64 publications

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“…Results have shown that xDNA forms stable, righthanded, sequence-selective helices, 4-8 and that individual xDNA bases in a template can encode successful formation of base pairs by DNA polymerase enzymes. 9 However, correct function in vitro is far from active functioning in a living cell, where many enzymes must operate simultaneously. To date, no unnatural genetic architectures have been used with general success to encode amino acids of a protein in a living system.…”

Section: Introductionmentioning

confidence: 99%

Encoding Phenotype in Bacteria with an Alternative Genetic Set

et al. 2011

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An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but with benzene added, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands, and were transfected into E. coli. Although yielding fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.

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Section: Introductionmentioning

confidence: 99%

Encoding Phenotype in Bacteria with an Alternative Genetic Set

et al. 2011

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“…This might be achieved by developing polymerases for unnatural base pair replication and transcription [77,78], and by scrutinizing the codon-anticodon interactions in unnatural base pair translation. Alternative unnatural base pair schemes have been reported, including size-expanded base pairs [79,80] and four-hydrogenbonded base pairs [81]. Although the fidelity and efficiency of these base pairs in polymerase reactions are currently low, these base pairs might be developed into an orthogonal system, complementary to the present unnatural base pairs.…”

Section: Five-year Viewmentioning

confidence: 98%

Unnatural base pair systems for sensing and diagnostic applications

Kimoto

Cox

Hirao

2011

Expert Review of Molecular Diagnostics

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Expansion of the genetic alphabet by an unnatural base pair system provides a platform for the site-specific, enzymatic incorporation of extra, functional components into nucleic acids. Recently, several unnatural base pairs that exhibit high fidelity and efficiency in PCR have been developed. Functional groups of interest, such as fluorescent dyes, can be linked to the unnatural bases, and the modified base substrates are site-specifically incorporated into nucleic acids by polymerases. Furthermore, unique unnatural base pairs between fluorophore and quencher base analogs have been developed for imaging PCR amplification and as molecular beacons. Here, we describe the recent progress in the development of unnatural base pairs that function in PCR amplification and their applications as sensing and diagnostic tools.

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“…For example, our laboratory demonstrated that xDNA can be replicated in vitro by some DNA polymerases. 48 Even more surprising was the recent observation that xDNA bases can correctly encode genetic information in a living cell. 49 …”

Section: Synthetic Biologymentioning

confidence: 99%

Chemistry of nucleic acids: impacts in multiple fields

Khakshoor

Kool

2011

Chem. Commun.

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Research in nucleic acids has made major advances in the past decade in multiple fields of science and technology. Here we discuss some of the most important findings in DNA and RNA research in the fields of biology, chemistry, biotechnology, synthetic biology, nanostructures and optical materials, with emphasis on how chemistry has impacted, and is impacted by, these developments. Major challenges ahead include the development of new chemical strategies that allow synthetically modified nucleic acids to enter into, and function in, living systems.

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Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs

Cited by 25 publications

References 64 publications

Encoding Phenotype in Bacteria with an Alternative Genetic Set

Encoding Phenotype in Bacteria with an Alternative Genetic Set

Unnatural base pair systems for sensing and diagnostic applications

Chemistry of nucleic acids: impacts in multiple fields

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