Nucleic Acids Research Advance Access published online on October 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp884
Molecular Biology |
Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence
1Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117510, 2Singapore-MIT Alliance, E4-04-10, 4 Engineering Drive 3, NUS, Singapore 117576, 3NUS Tissue Engineering Program, Life Science Institute, NUS, 117510, 4Molecular Hepatology, School of Medicine and Pharmacology, Faculty of Medicine, Dentistry and Health Sciences, The University of Western Australia, 5Department of Chemical and Biomolecular Engineering, Block E5, 4 Engineering Drive 4, National University of Singapore, Singapore 117576 and 6Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
*To whom correspondence should be addressed. Tel: +65 6516 7657, extn 5307 (DSO building); Fax: +65 6872 3069; Email: bierm{at}nus.edu.sg
Received August 19, 2009. Revised October 1, 2009. Accepted October 2, 2009.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First authors.