A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage.
Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.
Duss
Olivier
O
Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
Maris
Christophe
C
von Schroetter
Christine
C
Allain
Frédéric H-T
FH
D002851:Chromatography, High Pressure Liquid; D011088:DNA Ligases; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic; D022661:RNA, Untranslated / Q000737:chemistry; D016914:Ribonuclease H; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D022661:RNA, Untranslated; D012313:RNA; D016914:Ribonuclease H; D011088:DNA Ligases
J Biomol NMR. 2010 Jan;46(1):113-25
19789981
Methods Enzymol. 2005;394:399-416
15808230
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):44-8
8552656
Chembiochem. 2007 May 25;8(8):896-902
17440909
Nat Protoc. 2009;4(6):947-59
19478810
Annu Rev Biophys Biomol Struct. 2006;35:319-42
16689639
Nat Struct Biol. 2003 Dec;10(12):1033-8
14578934
Prog Nucleic Acid Res Mol Biol. 2008;82:147-97
18929141
EMBO J. 1995 Jan 16;14(2):368-76
7835347
Nat Struct Mol Biol. 2007 Sep;14(9):807-13
17704818
Curr Opin Struct Biol. 2007 Oct;17(5):603-16
17913493
RNA. 2007 Aug;13(8):1384-9
17548432
J Am Chem Soc. 2005 Aug 31;127(34):12035-45
16117544
Nucleic Acids Res. 2008 Aug;36(14):e89
18583361
Cell. 2009 Feb 20;136(4):577-80
19239877
RNA. 2005 Dec;11(12):1909-14
16251384
Nucleic Acids Res. 1990 Jan 11;18(1):201
1689817
FEBS Lett. 1987 May 11;215(2):327-30
2438160
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
12824337
J Am Chem Soc. 2006 Oct 18;128(41):13344-5
17031932
Biochemistry. 1990 Sep 18;29(37):8793-7
2176835
Methods Enzymol. 2000;317:109-23
10829275
Nat Struct Mol Biol. 2005 Mar;12(3):218-24
15723077
RNA. 1996 Mar;2(3):289-96
8608452
Curr Opin Microbiol. 2007 Apr;10(2):156-63
17383221
Methods Enzymol. 1995;261:300-22
8569501
Nucleic Acids Res. 2003 Aug 1;31(15):e82
12888534
Nat Protoc. 2007;2(9):2139-47
17853869
Int J Mol Sci. 2008 Jun;9(7):1214-40
19325801
Methods Enzymol. 2009;469:27-46
20946783
Nucleic Acids Res. 1996 Mar 1;24(5):977-8
8600468