Nuclear spin Nucleic acids

New techniques for data analysis and improvements in instrumentation have now made it possible to carry out stmctural and conformational studies of biopolymers including proteins, polysaccharides, and nucleic acids. NMR, which may be done on noncrystalline materials in solution, provides a technique complementary to X-ray diffraction, which requires crystals for analysis. One-dimensional NMR, as described to this point, can offer structural data for smaller molecules. But proteins and other biopolymers with large numbers of protons will yield a very crowded spectrum with many overlapping lines. In multidimensional NMR (2-D, 3-D, 4-D), peaks are spread out through two or more axes to improve resolution. The techniques of correlation spectroscopy (COSY), nuclear Overhausser effect spectroscopy (NOESY), and transverse relaxation-optimized spectroscopy (TROSY) depend on the observation that nonequivalent protons interact with each other. By using multiple-pulse techniques, it is possible to perturb one nucleus and observe the effect on the spin states of other nuclei. The availability of powerful computers and Fourier transform (FT) calculations makes it possible to elucidate structures of proteins up to 40,000 daltons in molecular mass and there is future promise for studies on proteins over 100,000... 

The common theme so far in our correlation experiments has been to allow spins to evolve during q under the influence of directly coupled nuclear spins. We have seen the power of COSY, HMQC, HMBC, and INADEQUATE to provide us with detailed structural information for ipsenol, caryophyllene oxide, and lactose. In this section, we will develop another method for showing correlations and apply it to molecules with distinct, isolated proton spin systems such as carbohydrates, peptides, and nucleic acids. 

Direct observation of very small dipolar couplings between distant protons in the overcrowded spectra of biological molecules represents a challenging task. Now Bax and co-workers have presented a method which, by the appropriate manipulation of the nuclear spin Hamiltonian, allows effective decoupling of all protons outside a spectral region of interest and facilitates observation of interactions between remote protons separated by distances of up to 12 A. The method has been applied to measure remote dipolar couplings in an unlabelled nucleic acid. 

Bo = 11.7T (v ( H) 500MHz) cover the frequency ranges of 7.5, 19, 8.2, and 1.1 kHz, respectively. In addition, a hyperfine spHtting and multiplet structure of resonance signals can be observed as a result of electron-mediated nuclear spin-spin scalar interactions. Both chemical shifts and scalar interactions reflect the chemical environment and provide a wealth of structure-related data. Since specific information about the individual atoms is available, NMR data can be successfully used to calculate a three-dimensional structure of RNA and DNA molecules. Fuelled by methodological developments during the past 10 years, NMR became an indispensable tool for structure elucidation of nucleic acids. At present (April 2003), almost 600 NMR structures of nucleic acids and/or protein/nucleic acid complexes can be found in the PDB database. 

Spin the buffy coat suspended in PBS at 1600g for 5 mm and remove the supernatant with a sterile pastet Resuspend the cells in GTC-ME, which causes proteins to dissolve, nuclear proteins to dissociate from nucleic acids owing to loss of secondary structure, and inactivation of RNAases The volume of GTC-ME is dependent on the cell-count of PBS-suspended cells for 1-2 x 10 , add GTC-ME to a final volume of 1 mL, for >2-4 x 10 add GTC-ME to a volume of 2 mL and for >4-6 X 10 cells add GTC-ME to a volume of 3 mL, and so on. Cells in GTC-ME may then be stored at 4°C until RNA extraction. 

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