NMR Structure Characterization of isotopically labeled DNA Oligonucleotides
EMSL Project ID
2328
Abstract
DNA structure determination by nuclear magnetic resonance (NMR) spectroscopy is an under-restrained problem in general. Isotopic-labeling considerably increases the number and type of NMR experiments that can be collected on DNA samples, and, thus increases the potential number and classes of restraints that can be applied in NMR-based DNA structure calculations. As a result, isotopic labeling allows DNA structures to be better determined by NMR methods and the DNA dynamics can be better characterized. Isotopic labeling is routinely employed for ribonucleic acids (RNAs) and proteins. However, uniform or specific labeling of synthetic DNAs has not yet become widespread due to intrinsic difficulty and cost. Isotopic labeling might be most valuable for characterizing the structure and dynamics of damaged-DNAs. To our knowledge, no NMR-based damaged DNA structures have been solved with the benefit of isotope labeling. The synthetic requirements for preparing DNA samples that contain both isotope labels and site-specific DNA lesions are great. Many DNA lesions can, however, be introduced into a sequence using a modified phosphoramidite together with automated solid-state synthesis. Combined uniform or specific isotope labeling and introduction of site-specific DNA lesions, in many cases, will require DNA synthesis using phosphoramidite chemistry. We have prepared two DNA oligonucleotides with alternating uniformly isotopically labeled nucleotide. We will make complete measurements of residual dipolar couplings and refined the structures of the oligos. One sample is 12 basepairs in length and contains a 8-oxoguanosine lesion. In order to determine a high quality structure needed to determine the effect of the 8-oxoguanosine lesion on the DNA structure, residual dipolar couplings will be measured to enable a high quality NMR structure determination. The second sample is 16 base pairs, CGAGGTTTAAACCTCG, and contains a TTTAAA segment. By gel retardation measurements, this sequence is unbent, however, it remains possible that the sequence contains compensating bends. In order to determine a reliable structure needed to address this fundamental question of how DNA sequence context determines DNA structure, residual dipolar coupling restraints will be measured and the structure refined. Primarily low field instrumentation will be used for these studies. All experiments will be carried out at 500 MHz. We therefore request 6 weeks of 500 MHz time. The residual dipolar couplings will be made for DNA aligned in filamentous phage particles.
Project Details
Project type
Capability Research
Start Date
2001-10-01
End Date
2002-11-18
Status
Closed
Released Data Link
Team
Principal Investigator