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Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

2007; Nature Portfolio; Volume: 2; Issue: 10 Linguagem: Inglês

10.1038/nprot.2007.308

1754-2189

Peter Z. Qin, Ian S. Haworth, Qi Cai, Ana Karin Kusnetzow, Gian Paola G. Grant, Eric A. Price, Glenna Z. Sowa, A.M. Popova, Bruno Herreros, Honghang He,

Advanced NMR Techniques and Applications

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron–electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.