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SPEKTROSKOPIA NMR W BADANIACH STRUKTUR. VLNYCH KWASÓW NUKLEINOWYCH CZĘŚĆ I 49

ABSTRACT

NMR spectroscopy is a powerful method that allows determination of the structure and dynamics of nucleic acids and their complexes in solution with atomie rcsolution. A major breakthro-ugh in the structure determination of nucleic acids by NMR was introduction of advanced and efficient methods for the labeling of RNA and DNA with ⁿC and ^l5N and de\*elopment ofmultidi-mensional, heteronuclear NMR techniąues analogous to those used in protein NMR spectroscopy.

The resonance assignment is a crucial step in the NMR study. A spectrum must be assigned before useful structural information can be extracted. The assignment of RNA is considerably morę difficult than for DNA of surular size. This is mainly due to the much narrower spectral dispersion of the H2\ H3\ H4’ and H575” ribose protons relative to DNA. 'The methodology for seąuential assignment of nucleic acids via homonuclear NMR techniąues relies on the assi miptinn of helical structure and therefore fails in the case of nonhelical structures, that is typical of RNA.

Development of ¹³C/¹⁵N labeling techniąues has afforded heteronuclear multidementional expe-riments that utilize the favorable properties of ¹³C and ^,SN nuclei such as large one- and two-bond heteronuclear scalar coupling constants and large Chemical shift dis-persion. These experiments provide inereased sensitivity of double and triple resonance experiments and help in overcoming the problem of severe spectral overlap.

Progress in novel NMR methods stimulated also a design of experiments for conformation-independent seąuential assignment. In nucleic acids, experiments that correlate base resonances among themseh es as well as with sugar resonances allow unambiguous spectral assignment for the structures, where the conventional NOE-based methods may not be applied. Assignments of highly overlapped sugar resonances are facilitated enormously by the application of correlated experiments based on ^l3C-¹³C transfer. Additionally, triple resonance experiments allow correlation of neighbo-ring nucleotides through the phosphodiester backbone.

The arsenał of existing methods in structure calculations of nucleic acids by NMR spectroscopy has recently been extended. For example, NMR methods have been developed to detect and measure scalar couplings via hydrogen bonds. The information about hydrogen bonds provides very useful restraints for structural determination, especially in case of noncanonical motifs.

Furthermore, the use of methods that introduce anisotropic environments for nucleic acids in solution allows the measurement of residual dipolar couplings (RDC). RDCs yield orientation, rather than distance-based constraints. The RDCs contain global structural information on nucleic acids that cannot be obtained by standard solution NMR techniąues. These constraints can both improve the local structure of nucleic acids and provide novel data on the global structure.

Another NMR techniąue, TROSY, has been introduced to effectively suppress transverse relaxation of ^!H-¹⁵N and ¹H-^I,C moieties. TROSY selects exclusively the narrowline of a ^!H-¹⁵N doublet or ^lH—^]l3C multiplet, yielding improved spectral resolution and inereased sensitivity of NMR experiment.

Recent advances in solution NMR techniąues provide tools for structural studies of large (> 30 residues) nucleic acids molecules.

Keywords: NMR spectroscopy, DNA and RNA, ¹³C/^1SN isotopic labeling, assignment strategy

Słowa kluczowe: spektroskopia NMR, DNA i RNA, znakowanie izotopowe ¹³C/¹⁵N, strategia przypisywania sygnałów