Characterization of sequence-specific oligodeoxyribonucleoside methylphosphonates and their interaction with rabbit globin mRNA

Akira Murakami, Kathleen Blake, Paul S. Miller

Research output: Contribution to journalArticle

Abstract

Oligodeoxynucleoside methylphosphonates, nucleic acid analogues that contain nonionic, 3′-5′-linked methylphosphonate internucleotide bonds, can be used to control mRNA function in living cells. In order to use analogues of defined sequence in biochemical and biological experiments, methods have been developed to characterize the chain length and sequence of oligodeoxyribonucleoside methylphosphonates and to study their interaction with mRNA. Methylphosphonate oligomers that terminate at the 5′ end with a 3′-5′ internucleotide phosphodiester bond are readily phosphorylated by polynucleotide kinase. Treatment of these 32P end labeled oligomers with aqueous piperidine randomly hydrolyzes the methylphosphonate linkage and upon gel electrophoresis produces a ladder of oligomers, which allows the chain length of the oligomer to be determined. The sequence of 32P end labeled oligonucleoside methylphosphonates can be determined by a modified chemical sequencing procedure. The interaction of the oligomers with rabbit globin mRNA was studied. The oligomers hybridize with mRNA in agarose gels. The stability of the hybrids increases with increasing chain length of the oligomer. The binding site of the oligomers on mRNA can be determined by using the oligomer as a primer for reverse transcriptase. The length of the resulting transcript is determined by polyacrylamide gel electrophoresis after removal of the methylphosphonate primer by treatment with piperidine. The results indicate that binding and priming ability of the oligonucleoside methylphosphonates are affected by the secondary structure of the mRNA.

Original languageEnglish (US)
Pages (from-to)4041-4046
Number of pages6
JournalBiochemistry®
Volume24
Issue number15
StatePublished - 1985

Fingerprint

Globins
Oligomers
Rabbits
Messenger RNA
Chain length
Gels
Polynucleotide 5'-Hydroxyl-Kinase
Electrophoresis
RNA-Directed DNA Polymerase
Sepharose
Nucleic Acids
dideoxyribonucleoside methylphosphonates
methylphosphonic acid
Polyacrylamide Gel Electrophoresis
Binding Sites
Ladders
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of sequence-specific oligodeoxyribonucleoside methylphosphonates and their interaction with rabbit globin mRNA. / Murakami, Akira; Blake, Kathleen; Miller, Paul S.

In: Biochemistry®, Vol. 24, No. 15, 1985, p. 4041-4046.

Research output: Contribution to journalArticle

Murakami, Akira ; Blake, Kathleen ; Miller, Paul S. / Characterization of sequence-specific oligodeoxyribonucleoside methylphosphonates and their interaction with rabbit globin mRNA. In: Biochemistry®. 1985 ; Vol. 24, No. 15. pp. 4041-4046.
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