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Efficient nucleic acid detection by templated reductive quencher release.

ABSTRACT: RNA-templated fluorescence activation is a nucleic acid detection strategy that offers the possibility of direct visual detection of genetic information in living cells. Here we describe a new reaction strategy for fluorescence activation in which a phosphine on one DNA probe reduces an azide group in a linker on a second probe, resulting in linker cleavage and release of a fluorescence quenching group. These "Q-STAR" probes are shown to yield a strong fluorescence turn-on signal in approximately 20 min, with very low background and substantial amplification by turnover on the template. A green/red pair of such probes allowed the discrimination of two bacterial species by a single nucleotide difference in their 16S rRNA. The beneficial properties of the reductive quencher release design make these probes promising candidates for widespread application in the detection of nucleic acids in vitro and in cells.

SUBMITTER: Franzini RM

PROVIDER: S-EPMC2774910 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Efficient nucleic acid detection by templated reductive quencher release.

Franzini Raphael M RM Kool Eric T ET

Journal of the American Chemical Society 20091101 44

RNA-templated fluorescence activation is a nucleic acid detection strategy that offers the possibility of direct visual detection of genetic information in living cells. Here we describe a new reaction strategy for fluorescence activation in which a phosphine on one DNA probe reduces an azide group in a linker on a second probe, resulting in linker cleavage and release of a fluorescence quenching group. These "Q-STAR" probes are shown to yield a strong fluorescence turn-on signal in approximatel ...[more]

PMID: 19886694

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Project description:The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.

Dataset Information

Efficient nucleic acid detection by templated reductive quencher release.

Publications

Efficient nucleic acid detection by templated reductive quencher release.

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