Unnatural base pair systems for sensing and diagnostic applications

Kimoto, Michiko; Cox, Robert Sidney; Hirao, Ichiro

doi:10.1586/erm.11.5

Cited by 52 publications

(37 citation statements)

References 79 publications

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“…Today, a few groups have begun to take the next step (8–11), adding replicable nucleotides to the DNA alphabet, creating a new molecular biology to support this expanded laboratory in vitro evolution (LIVE) (12), and developing the analytical chemical tools needed to sequence expanded DNA survivors that might emerge under selective pressure (13). Of particular promise are artificially expanded genetic information systems (AEGIS), which shuffle hydrogen-bonding groups within a Watson–Crick geometry to generate additional replicable nucleotides (14).…”

Section: Introductionmentioning

confidence: 99%

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

et al. 2016

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Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.

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Section: Introductionmentioning

confidence: 99%

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

et al. 2016

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“…In addition, the attachment of functional groups to unnatural bases enables the site-specific functionalization of DNA and RNA molecules by replication and transcription (10–16). The capabilities of several unnatural base pairs have been demonstrated, and they have been utilized in diagnostics for target DNA detection and in basic research for site-specific labeling of nucleic acid molecules (9,17–21). For example, an unnatural base pair between isoguanine and isocytosine (22) is available as Plexor, in a multiplex real-time quantitative PCR detection platform (23–25).…”

Section: Introductionmentioning

confidence: 99%

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Yamashige

Kimoto

Takezawa

et al. 2011

Nucleic Acids Research

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Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds–Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds–Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds–Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds–Px pair system provides a framework for new biotechnology.

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“…The realization of this genetic expansion system requires the development of an unnatural base pair that functions in biological systems, such as replication, transcription, and translation, with highly exclusive selectivity as a third base pair, along with the natural A-T and G-C pairs. Researchers are attempting to create expanded systems, and many unnatural base pairs have been designed and tested in in vitro biological systems [1][2][3][4][5]. Among them, some unnatural base pairs have exhibited high selectivity as a third base pair in PCR amplification and/or transcription [6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

et al. 2012

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Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo [4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

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Unnatural base pair systems for sensing and diagnostic applications

Cited by 52 publications

References 79 publications

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

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