title: Preparation of modified long-mer RNAs and analysis of FMN binding to the ypaA aptamer from B. subtilis.
abstract: In recent years, RNA has been shown to fulfil a number of cellular functions. This has led to much interest in elucidation of the structure of functional RNA molecules, and thus, in the preparation of suitably functionalized RNAs. The chemical synthesis of RNAs allows for the site-specific modification; however, is limited to sequences of about 60-70 nucleotides in length. At the example of the flavine mononucleotide (FMN) responsive aptamer of the ypaA riboswitch from B. subtilis, we demonstrate the highly efficient preparation of site-specifically modified long-mer RNAs. Our strategy consists of the chemical synthesis of fragments followed by enzymatic or chemical ligation. Splint ligation with T4 RNA ligase turned out to be most successful among the enyzymatic protocols. Highly efficient chemical ligation was performed by azide-alkyne cycloaddition of suitably modified RNA fragments. Wild-type and 2-aminopurine (2-AP)-modified variants of the ypaA aptamer were prepared. FMN binding to all synthesized ypaA aptamer variants is demonstrated. However, dissociation of FMN from its binding site by reduction of the isoalloxazin unit as demonstrated before for a small-hairpin-derived aptazyme could not be shown. This implies that either FMN is less accessible to reduction when it is bound to its natural aptamer; that reduced FMN remains bound to the aptamer; or that FMN upon reduction indeed is released from its binding site, without the aptamer folding back in the natural ligand-free state. The results of this study are of general interest to the preparation of site-specifically modified RNAs for investigation into structure and function.
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Frommer
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Hieronymus
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany; PG and Research Department of Chemistry; Thiagarajar College (Autonomous); Madurai, India.
authors.forename: Tamil
authors.initials: T
authors.lastname: Selvi Arunachalam
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany; Hochschule Neubrandenburg; Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften; AG Landwirtschaftliche Chemie; Neubrandenburg, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Heeren
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany; Friedrich-Loeffler-Institut; Institut für Virusdiagnostik; Südufer 10; Riems, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Jenckel
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Strahl
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Appel
authors.affiliation: Ernst Moritz Arndt University Greifswald; Institute for Biochemistry; Greifswald, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Müller
mesh_terms: D000494:Allosteric Regulation; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D057930:Click Chemistry; D005486:Flavin Mononucleotide / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism; D058928:Riboswitch
chemical_list: D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; D058928:Riboswitch; D012313:RNA; D005486:Flavin Mononucleotide
keywords: 2-aminopurine; Click chemistry; RNA; aptamer; fluorescence; ligation; riboswitch; synthesis
references.citation: J Org Chem. 2002 May 3;67(9):3057-64
references.pmid: 11975567
references.citation: Angew Chem Int Ed Engl. 2010 Oct 4;49(41):7470-2
references.pmid: 21038451
references.citation: Top Curr Chem. 2012;321:159-98
references.pmid: 22160388
references.citation: Nat Rev Microbiol. 2012 Mar 16;10(4):255-65
references.pmid: 22421878
references.citation: Annu Rev Biophys. 2008;37:117-33
references.pmid: 18573075
references.citation: Bioconjug Chem. 2014 Jan 15;25(1):188-95
references.pmid: 24358989
references.citation: Chembiochem. 2005 Dec;6(12):2158-62
references.pmid: 16276501
references.citation: Angew Chem Int Ed Engl. 2002 Jul 15;41(14):2596-9
references.pmid: 12203546
references.citation: Nature. 2009 Mar 12;458(7235):233-7
references.pmid: 19169240
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15908-13
references.pmid: 12456892
references.citation: Cold Spring Harb Perspect Biol. 2012 Feb 01;4(2):
references.pmid: 21106649
references.citation: FEBS Lett. 1988 May 9;232(1):153-5
references.pmid: 2835262
references.citation: Angew Chem Int Ed Engl. 2009;48(22):4056-60
references.pmid: 19396850
references.citation: Bioorg Med Chem. 2013 Oct 15;21(20):6171-80
references.pmid: 23664496
references.citation: Chem Soc Rev. 2010 Apr;39(4):1388-405
references.pmid: 20309492
references.citation: Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):E3256-64
references.pmid: 23940363
references.citation: Curr Opin Struct Biol. 2012 Jun;22(3):279-86
references.pmid: 22579413
references.citation: J Photochem Photobiol B. 2007 Apr 2;87(1):37-48
references.pmid: 17292618
references.citation: Methods Mol Biol. 2012;848:41-59
references.pmid: 22315062
references.citation: Nucleic Acids Res. 1988 May 11;16(9):3721-38
references.pmid: 3375071
references.citation: Nucleic Acids Res. 2002 Jul 15;30(14):3141-51
references.pmid: 12136096
references.citation: Cell. 2002 Nov 27;111(5):747-56
references.pmid: 12464185
references.citation: Trends Genet. 1999 Nov;15(11):439-42
references.pmid: 10529804
references.citation: Biochemistry. 1984 Oct 9;23(21):4859-67
references.pmid: 6498164
references.citation: Acc Chem Res. 2009 Oct 20;42(10):1521-31
references.pmid: 19572701
references.citation: J Am Chem Soc. 2007 May 30;129(21):6859-64
references.pmid: 17488075
references.citation: Wiley Interdiscip Rev RNA. 2012 May-Jun;3(3):369-84
references.pmid: 21957061
references.citation: Curr Opin Struct Biol. 2010 Jun;20(3):295-304
references.pmid: 20447823
references.citation: J Am Chem Soc. 2009 Mar 25;131(11):3958-64
references.pmid: 19292490
references.citation: Mol Microbiol. 2011 Jun;80(5):1148-54
references.pmid: 21477128
references.citation: Biochemistry. 1984 Oct 9;23(21):4868-72
references.pmid: 6541948
references.citation: Nucleic Acids Res. 2007;35(15):5262-73
references.pmid: 17686787
references.citation: Tetrahedron Lett. 1997 Aug 11;38(32):5595-5598
references.pmid: 19924262
references.citation: J Nucleic Acids. 2011;2011:805253
references.pmid: 22013508
references.citation: Angew Chem Int Ed Engl. 2006 Mar 20;45(13):2127-9
references.pmid: 16502442
references.citation: Org Lett. 2004 Aug 19;6(17):2853-5
references.pmid: 15330631
references.citation: Cell. 2013 Jan 17;152(1-2):17-24
references.pmid: 23332744
references.citation: J Am Chem Soc. 2011 Mar 2;133(8):2463-80
references.pmid: 21291253
references.citation: Angew Chem Int Ed Engl. 2003 May 30;42(21):2424-7
references.pmid: 12783515
references.citation: Chembiochem. 2011 Jan 3;12(1):125-31
references.pmid: 21132831
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15329-34
references.pmid: 20713730
references.citation: Nature. 2013 Jul 18;499(7458):355-9
references.pmid: 23842498