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Quencher-free multiplexed monitoring of DNA reaction circuits.

ABSTRACT: We present a simple yet efficient technique to monitor the dynamics of DNA-based reaction circuits. This technique relies on the labeling of DNA oligonucleotides with a single fluorescent modification. In this quencher-free setup, the signal is modulated by the interaction of the 3'-terminus fluorophore with the nucleobases themselves. Depending on the nature of the fluorophore's nearest base pair, fluorescence intensity is decreased or increased upon hybridization. By tuning the 3'-terminal nucleotides, it is possible to obtain opposite changes in fluorescence intensity for oligonucleotides whose hybridization site is shifted by a single base. Quenching by nucleobases provides a highly sequence-specific monitoring technique, which presents a high sensitivity even for small oligonucleotides. Compared with other sequence-specific detection methods, it is relatively non-invasive and compatible with the complex dynamics of DNA reaction circuits. As an application, we show the implementation of nucleobase quenching to monitor a DNA-based chemical oscillator, allowing us to follow in real time and quantitatively the dephased oscillations of the components of the network. This cost-effective monitoring technique should be widely implementable to other DNA-based reaction systems.

SUBMITTER: Padirac A

PROVIDER: S-EPMC3424586 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Quencher-free multiplexed monitoring of DNA reaction circuits.

Padirac Adrien A Fujii Teruo T Rondelez Yannick Y

Nucleic acids research 20120629 15

We present a simple yet efficient technique to monitor the dynamics of DNA-based reaction circuits. This technique relies on the labeling of DNA oligonucleotides with a single fluorescent modification. In this quencher-free setup, the signal is modulated by the interaction of the 3'-terminus fluorophore with the nucleobases themselves. Depending on the nature of the fluorophore's nearest base pair, fluorescence intensity is decreased or increased upon hybridization. By tuning the 3'-terminal nuc ...[more]

PMID: 22753028

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Project description:Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.

Dataset Information

Quencher-free multiplexed monitoring of DNA reaction circuits.

Publications

Quencher-free multiplexed monitoring of DNA reaction circuits.

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