title: Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.
abstract: We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.
authors.affiliation: Department of Chemistry, School of Arts and Sciences 231 South 34th Street, Philadelphia, PA 19104, USA.
authors.forename: Lida K
authors.initials: LK
authors.lastname: Gifford
authors.forename: Joanna B
authors.initials: JB
authors.lastname: Opalinska
authors.forename: David
authors.initials: D
authors.lastname: Jordan
authors.forename: Vikram
authors.initials: V
authors.lastname: Pattanayak
authors.forename: Paul
authors.initials: P
authors.lastname: Greenham
authors.forename: Anna
authors.initials: A
authors.lastname: Kalota
authors.forename: Michelle
authors.initials: M
authors.lastname: Robbins
authors.forename: Kathy
authors.initials: K
authors.lastname: Vernovsky
authors.forename: Lesbeth C
authors.initials: LC
authors.lastname: Rodriguez
authors.forename: Bao T
authors.initials: BT
authors.lastname: Do
authors.forename: Ponzy
authors.initials: P
authors.lastname: Lu
authors.forename: Alan M
authors.initials: AM
authors.lastname: Gewirtz
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry*; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051560:Proto-Oncogene Proteins c-bcl-6; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D020319:Oligodeoxyribonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D051560:Proto-Oncogene Proteins c-bcl-6; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D014157:Transcription Factors; D019009:Proto-Oncogene Proteins c-kit
references.citation: Stem Cells. 2000;18(5):307-19
references.pmid: 11007915
references.citation: Science. 1999 Sep 10;285(5434):1685
references.pmid: 10523186
references.citation: Pharmacol Rev. 2000 Sep;52(3):325-47
references.pmid: 10977866
references.citation: Curr Opin Mol Ther. 2000 Jun;2(3):297-303
references.pmid: 11249624
references.citation: Anal Chem. 2001 Nov 15;73(22):5544-50
references.pmid: 11816586
references.citation: Nat Rev Mol Cell Biol. 2002 Mar;3(3):195-205
references.pmid: 11994740
references.citation: Nature. 2002 Jul 11;418(6894):244-51
references.pmid: 12110901
references.citation: Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7
references.pmid: 14769924
references.citation: Eur J Biochem. 1976 AUG 1;67(1):247-56
references.pmid: 823012
references.citation: EMBO J. 1987 Nov;6(11):3341-51
references.pmid: 2448137
references.citation: Science. 1988 Dec 2;242(4883):1303-6
references.pmid: 2461588
references.citation: Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2351-5
references.pmid: 2006173
references.citation: Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2702-6
references.pmid: 1849273
references.citation: Cell. 1991 May 17;65(4):677-89
references.pmid: 1709592
references.citation: Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1710-4
references.pmid: 1371882
references.citation: Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5262-6
references.pmid: 8506375
references.citation: Blood. 1994 Sep 1;84(5):1361-92
references.pmid: 8068936
references.citation: Appl Biochem Biotechnol. 1995 Jul-Sep;54(1-3):57-61
references.pmid: 7486985
references.citation: EMBO J. 1995 Dec 15;14(24):6209-17
references.pmid: 8557040
references.citation: Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6947-52
references.pmid: 8692924
references.citation: Nat Biotechnol. 1996 Mar;14(3):303-8
references.pmid: 9630890
references.citation: Blood. 1998 Aug 1;92(3):712-36
references.pmid: 9680338
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11538-43
references.pmid: 9751701
references.citation: J Mol Biol. 1999 Oct 22;293(2):271-81
references.pmid: 10550208