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Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.

ABSTRACT: DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity.

SUBMITTER: Suspene R

PROVIDER: S-EPMC2475633 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.

Suspène Rodolphe R Renard Myrtille M Henry Michel M Guétard Denise D Puyraimond-Zemmour David D Billecocq Agnès A Bouloy Michèle M Tangy Frederic F Vartanian Jean-Pierre JP Wain-Hobson Simon S

Nucleic acids research 20080530 12

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR ...[more]

PMID: 18515351

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Project description:We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%–8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.

Dataset Information

Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.

Publications

Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs.

Similar Datasets

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