Journal
CHEMBIOCHEM
Volume 8, Issue 16, Pages 1957-1964Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200700245
Keywords
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Funding
- Engineering and Physical Sciences Research Council [GR/S85719/01] Funding Source: researchfish
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Many biological systems, especially those based on nucleic acids, ore structurally heterogeneous in solution. We demonstrate here the ability to measure multiple distances, of between 2 and 7 nm, from a heterogeneous mixture of double-spin-labeled DNA duplexes. We have constructed a DNA distance ruler based on the attachment of nitroxide spin labels to 2'-amino-modified nucleosides. The distribution of distances between the spin labels was obtained by Tikhonov regularization analysis of the dipolar coupling evolution data measured by using the electron para-magnetic resonance method, pulsed-electron double resonance (PELDOR). Optimization of the conditions and techniques used in the preparation of the samples has allowed us to increase the sensitivity and reduce aggregation artifacts. As a result, we have been able to demonstrate deconvolution of distances from structurally heterogeneous samples and show the limits of the technique by examining data derived from up to five DNA duplexes, in a single mixture, in which the concentration of each species was as low as 5 mu M.
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