Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments.
The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studied by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1' and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RN oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1' and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.
Sklenár
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V
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Peterson
R D
RD
Rejante
M R
MR
Feigon
J
J
D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry; D015694:Gene Products, rev / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry; D009587:Nitrogen Isotopes; D009711:Nucleotides / Q000737:chemistry*; D009843:Oligoribonucleotides / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D002241:Carbohydrates; D015694:Gene Products, rev; D006027:Glycosides; D009587:Nitrogen Isotopes; D009711:Nucleotides; D009843:Oligoribonucleotides; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D006859:Hydrogen; D009584:Nitrogen