An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction.
An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked to a streptavidin-binding aptamer that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding aptamer is only activated by an editing-induced conformational change. An electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis, and binding affinity measurements were used to characterize the reporter. The highly sensitive assay could be adapted to a broad range of RNA processing reactions.
Liang
Shuang
S
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Connell
Gregory J
GJ
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D004563:Electrochemistry; D049449:Luminescence; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012689:Sequence Homology, Nucleic Acid
D052157:Aptamers, Nucleotide
J Clin Invest. 2008 Apr;118(4):1301-10
18382742
Methods Enzymol. 1989;180:212-27
2515419
J Biol Chem. 1999 Mar 5;274(10):6295-304
10037718
EMBO J. 1996 Dec 2;15(23):6758-65
8978701
RNA. 2006 Jun;12(6):1038-49
16611942
Methods Enzymol. 1989;180:51-62
2482430
Cell. 1986 Sep 12;46(6):819-26
3019552
Proc Natl Acad Sci U S A. 2008 Nov 11;105(45):17278-83
18981420
Nature. 1990 Aug 30;346(6287):818-22
1697402
J Biol Chem. 2000 Oct 27;275(43):33911-9
10940300
Methods Enzymol. 1996;267:315-35
8743325
Mol Cell Biol. 2002 Mar;22(5):1567-76
11839822
Biochemistry. 1999 Mar 9;38(10):3168-74
10074372
RNA. 2001 Apr;7(4):632-41
11345441
EMBO J. 1998 Nov 2;17(21):6368-76
9799244
Science. 1992 May 15;256(5059):992-7
1589782
EMBO J. 1997 Jul 1;16(13):4069-81
9233816
RNA. 2003 Apr;9(4):469-83
12649498
Science. 1990 Aug 3;249(4968):505-10
2200121
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706-10
2479010
Science. 1996 Aug 30;273(5279):1189-95
8703045
Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
12824337
Mol Cell Biol. 2008 Jan;28(1):122-30
17954557
Cell. 1990 Jan 26;60(2):189-98
1688737
J Biol Chem. 2003 Dec 19;278(51):51167-75
14532294
RNA. 2002 Apr;8(4):548-54
11991648
Science. 2001 Mar 16;291(5511):2159-62
11251122
Comb Chem High Throughput Screen. 2002 Jun;5(4):263-70
12052178
J Pharmacol Exp Ther. 2005 May;313(2):902-8
15743924
Cell. 1996 Mar 22;84(6):831-41
8601307
Mol Cell Biol. 2001 Jan;21(2):380-9
11134327
EMBO J. 2003 Feb 17;22(4):913-24
12574127
RNA. 1999 Sep;5(9):1268-72
10496227