Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.
RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-kappaB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-kappaB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-kappaB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-kappaB p50(2) and p65(2), respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-kappaB p65(2) in vitro.
Wurster
Susan E
SE
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Bida
John Paul
JP
Her
Yeng F
YF
Maher
L James
LJ
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D016296:Mutagenesis; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors; D009690:Nucleic Acid Conformation; D055503:Protein Multimerization; D052156:SELEX Aptamer Technique; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D052157:Aptamers, Nucleotide; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA
RNA. 2007 Apr;13(4):614-22
17283213
RNA. 2008 Jun;14(6):1154-63
18441054
Mol Carcinog. 1999 Oct;26(2):119-29
10506755
Ann Surg. 2004 Apr;239(4):491-500
15024310
Oncogene. 2003 Jan 9;22(1):50-8
12527907
Genes Dev. 1995 Nov 15;9(22):2723-35
7590248
Nat Immunol. 2002 Mar;3(3):221-7
11875461
Biochemistry. 2001 Feb 27;40(8):2433-8
11327864
Anal Biochem. 2002 Jul 15;306(2):290-7
12123668
Methods Enzymol. 1999;306:93-113
10432449
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3930-5
12637683
Mol Cell Biol. 2000 Mar;20(5):1626-38
10669740
Curr Opin Struct Biol. 2004 Feb;14(1):21-7
15102445
J Clin Invest. 2005 Oct;115(10):2625-32
16200195
Oncogene. 1995 Sep 7;11(5):999-1003
7675461
Mol Cell Biol. 1993 Sep;13(9):5141-8
8355674
Cancer Lett. 2001 Oct 10;171(2):165-72
11520600
J Mol Biol. 1999 Jun 18;289(4):1029-40
10369780
J Biol Chem. 2001 Jun 1;276(22):18934-40
11259436
Clin Cancer Res. 2007 Feb 15;13(4):1076-82
17317814
Oncogene. 1999 Nov 22;18(49):6845-52
10602460
Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9268-73
12886018
Biochem Biophys Res Commun. 2007 Aug 3;359(3):475-80
17560552
J Clin Invest. 1997 Dec 15;100(12):2952-60
9399940
J Clin Invest. 1997 Dec 15;100(12):2961-9
9399941
Prostate. 2002 Aug 1;52(3):183-200
12111695
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8496-501
8710898
Genes Dev. 2004 Sep 15;18(18):2195-224
15371334
RNA. 1999 Apr;5(4):596-601
10199575
Clin Cancer Res. 1999 Jan;5(1):119-27
9918209
Oncogene. 2006 Oct 30;25(51):6800-16
17072329
Cancer Cell. 2004 Nov;6(5):471-83
15542431
Anticancer Res. 2004 Mar-Apr;24(2B):675-81
15161011
RNA. 2008 Jun;14(6):1037-47
18426920
Cancer Res. 2001 May 1;61(9):3810-8
11325857
FEBS Lett. 2009 May 6;583(9):1415-21
19358846
Nucleic Acids Res. 2006 Aug 07;34(13):3755-61
16893958
Genes Dev. 1992 May;6(5):775-87
1577272
Biochemistry. 2002 Jul 30;41(30):9696-706
12135392
J Cell Sci. 2002 Jan 1;115(Pt 1):141-51
11801732
Biochemistry. 1999 Mar 9;38(10):3168-74
10074372