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    Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

    195533_103599_Lareu_NucAcidsRes_2010.pdf (2.739Mb)
    Access Status
    Open access
    Authors
    Harve, K.
    Lareu, Ricky R.
    Rajagopalan, R.
    Raghunath, M.
    Date
    2010
    Type
    Journal Article
    
    Metadata
    Show full item record
    Citation
    Harve, Karthik S. and Lareu, Ricky and Rajagopalan, Raj and Raghunath, Michael. 2010. Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence. Nucleic Acids Research. 38 (1): pp. 172-181.
    Source Title
    Nucleic Acids Research
    DOI
    10.1093/nar/gkp884
    ISSN
    0305-1048
    Remarks

    This article is published under the Open Access publishing model and distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by-nc/2.5/. Please refer to the licence to obtain terms for any further reuse or distribution of this work.

    URI
    http://hdl.handle.net/20.500.11937/30252
    Collection
    • Curtin Research Publications
    Abstract

    Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.

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