Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.
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.
Gifford
Lida K
LK
Department of Chemistry, School of Arts and Sciences 231 South 34th Street, Philadelphia, PA 19104, USA.
Opalinska
Joanna B
JB
Jordan
David
D
Pattanayak
Vikram
V
Greenham
Paul
P
Kalota
Anna
A
Robbins
Michelle
M
Vernovsky
Kathy
K
Rodriguez
Lesbeth C
LC
Do
Bao T
BT
Lu
Ponzy
P
Gewirtz
Alan M
AM
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
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
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