Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence
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...
| Main Authors: | , , , |
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| Format: | Journal Article |
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Oxford University Press
2010
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| Online Access: | http://hdl.handle.net/20.500.11937/30252 |
| _version_ | 1848753035170283520 |
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| author | Harve, K. Lareu, Ricky R. Rajagopalan, R. Raghunath, M. |
| author_facet | Harve, K. Lareu, Ricky R. Rajagopalan, R. Raghunath, M. |
| author_sort | Harve, K. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T08:18:06Z |
| format | Journal Article |
| id | curtin-20.500.11937-30252 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T08:18:06Z |
| publishDate | 2010 |
| publisher | Oxford University Press |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-302522017-09-13T15:31:15Z Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence Harve, K. Lareu, Ricky R. Rajagopalan, R. Raghunath, M. 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. 2010 Journal Article http://hdl.handle.net/20.500.11937/30252 10.1093/nar/gkp884 Oxford University Press fulltext |
| spellingShingle | Harve, K. Lareu, Ricky R. Rajagopalan, R. Raghunath, M. Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title | Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title_full | Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title_fullStr | Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title_full_unstemmed | Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title_short | Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence |
| title_sort | understanding how the crowded interior of cells stabilizes dna/dna and dna/rna hybrids–in silico predictions and in vitro evidence |
| url | http://hdl.handle.net/20.500.11937/30252 |