A mutate-and-map strategy accurately infers the base pairs of a 35-nucleotide model RNA.
We present a rapid experimental strategy for inferring base pairs in structured RNAs via an information-rich extension of classic chemical mapping approaches. The mutate-and-map method, previously applied to a DNA/RNA helix, systematically searches for single mutations that enhance the chemical accessibility of base-pairing partners distant in sequence. To test this strategy for structured RNAs, we have carried out mutate-and-map measurements for a 35-nt hairpin, called the MedLoop RNA, embedded within an 80-nt sequence. We demonstrate the synthesis of all 105 single mutants of the MedLoop RNA sequence and present high-throughput DMS, CMCT, and SHAPE modification measurements for this library at single-nucleotide resolution. The resulting two-dimensional data reveal visually clear, punctate features corresponding to RNA base pair interactions as well as more complex features; these signals can be qualitatively rationalized by comparison to secondary structure predictions. Finally, we present an automated, sequence-blind analysis that permits the confident identification of nine of the 10 MedLoop RNA base pairs at single-nucleotide resolution, while discriminating against all 1460 false-positive base pairs. These results establish the accuracy and information content of the mutate-and-map strategy and support its feasibility for rapidly characterizing the base-pairing patterns of larger and more complex RNA systems.
Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
Wipapat
W
Kladwang
Pablo
P
Cordero
Rhiju
R
Das
D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics
D012313:RNA; D004247:DNA
Curr Opin Struct Biol. 2006 Jun;16(3):270-8
16713706
Nat Methods. 2010 Apr;7(4):291-4
20190761
RNA. 2006 Aug;12(8):1475-85
16809815
Free Radic Res Commun. 1991;12-13 Pt 2:521-9
1648010
Biochemistry. 2007 Nov 20;46(46):13425-36
17958380
Curr Opin Struct Biol. 2001 Feb;11(1):70-82
11179895
Nature. 2006 Sep 14;443(7108):167-72
16915236
Biochemistry. 2000 Sep 26;39(38):11748-62
10995243
RNA. 2000 Feb;6(2):220-32
10688361
RNA. 1996 Apr;2(4):367-79
8634917
Science. 2005 Sep 2;309(5740):1508-14
16141058
Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11660-4
7526401
RNA. 2010 Jun;16(6):1108-17
20413617
Methods Enzymol. 1989;180:212-27
2515419
Science. 2008 Mar 28;319(5871):1787-9
18369136
Nat Protoc. 2006;1(3):1610-6
17406453
Nucleic Acids Res. 2011 Feb;39(3):1081-94
20876693
Proc Natl Acad Sci U S A. 1994 May 24;91(11):4955-9
8197164
Angew Chem Int Ed Engl. 2003 Jul 28;42(29):3340-63
12888958
Chem Biol. 1996 Dec;3(12):993-1009
9000010
Proc Natl Acad Sci U S A. 2004 May 11;101(19):7287-92
15123812
J Mol Biol. 1999 Jan 8;285(1):97-105
9878391
Biochemistry. 2010 Oct 12;49(40):8669-79
20681613
Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6421-6
15096624
RNA. 2005 Mar;11(3):344-54
15701734
Annu Rev Biophys Biomol Struct. 2006;35:319-42
16689639
Curr Opin Struct Biol. 2002 Jun;12(3):296-300
12127447
Nature. 1992 Jul 9;358(6382):123-8
1377367
J Mol Biol. 1999 May 21;288(5):911-40
10329189
Adv Protein Chem. 1981;34:167-339
7020376
Biochemistry. 2008 Aug 19;47(33):8504-13
18642882
RNA. 2008 Sep;14(9):1845-51
18658118
Acc Chem Res. 2001 Oct;34(10):836-43
11601968
Fold Des. 1997;2(5):295-306
9377713
Nucleic Acids Res. 1992 Nov 11;20(21):5785-95
1454539
Biochemistry. 2010 Sep 7;49(35):7414-6
20677780
Cell. 2009 Feb 20;136(4):604-9
19239882
Cell. 2002 Oct 4;111(1):129-40
12372306
Nucleic Acids Res. 2006 May 08;34(8):e64
16682443
Curr Protoc Bioinformatics. 2004 Feb;Chapter 12:Unit 12.2
18428716
Biochem Soc Trans. 2008 Aug;36(Pt 4):567-74
18631119
Nat Protoc. 2007;2(10):2608-23
17948004
Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033-7
3277187
Nature. 1969 Nov 22;224(5221):759-63
5361649
Biochemistry. 1999 Oct 26;38(43):14214-23
10571995
PLoS Biol. 2008 Apr 29;6(4):e96
18447581
Science. 1989 Jun 2;244(4908):1081-5
2471267
Proc Natl Acad Sci U S A. 1979 May;76(5):2232-5
287063
Nat Protoc. 2008;3(5):811-23
18451789
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4144-9
18322008
Nucleic Acids Res. 2008 Jun;36(11):e63
18477638
Cell. 1998 Feb 6;92(3):315-26
9476892
Proc Natl Acad Sci U S A. 2009 Jan 6;106(1):97-102
19109441
Biochemistry. 2008 Sep 23;47(38):10178-87
18754680
Nat Struct Biol. 1998 Dec;5(12):1037-42
9846872
Methods Enzymol. 2000;318:446-60
10890005
J Mol Biol. 2006 Jun 9;359(3):526-32
16500677
Proc Natl Acad Sci U S A. 1980 Aug;77(8):4679-82
6159633
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8950-4
10908667
RNA. 2008 Oct;14(10):1979-90
18772246
Bioinformatics. 2009 Aug 1;25(15):1974-5
19398448
PLoS One. 2008;3(10):e3229
18927606
Nature. 2009 Aug 6;460(7256):711-6
19661910
FEBS Lett. 1985 Jan 7;179(2):187-98
2981703
Biopolymers. 1999-2000;52(4):157-67
11295748