Nucleic acid structure determination

Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes. X-ray crystallography is not common for nucleic acids alone, since neither DNA nor RNA readily form crystals. This is due to the greater degree of intrinsic disorder and dynamism in nucleic acid structures and the negatively charged (deoxy)ribose-phosphate backbones, which repel each other in close proximity. Therefore, crystallized nucleic acids tend to be complexed with a protein of interest to provide structural order and neutralize the negative charge. Nuclear magnetic resonance spectroscopy of nucleic acids Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. As of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy. Nucleic acid NMR uses similar techniques as protein NMR, but has several differences. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR, and because nucleic acid double helices are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are 1H or proton NMR, 13C NMR, 15N NMR, and 31P NMR. Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space.