Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells

Joan C. Politz, Krishan L. Taneja, Robert H. Singer

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18 h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.

Original languageEnglish (US)
Pages (from-to)4946-4953
Number of pages8
JournalNucleic Acids Research
Volume23
Issue number24
StatePublished - Dec 25 1995
Externally publishedYes

Fingerprint

Oligonucleotides
Oligodeoxyribonucleotides
RNA
Assays
Transcription
Cells
Cell
Fluorescent Dyes
Messenger RNA
Actins
Antisense Oligonucleotides
Cell culture
Detergents
Complementary DNA
Actin
Tissue
Reverse Transcription
Molecules
oligo (dT)
Hybridization

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells. / Politz, Joan C.; Taneja, Krishan L.; Singer, Robert H.

In: Nucleic Acids Research, Vol. 23, No. 24, 25.12.1995, p. 4946-4953.

Research output: Contribution to journalArticle

@article{337c01d25b0a47df9b7ac060f288096d,
title = "Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells",
abstract = "Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18 h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.",
author = "Politz, {Joan C.} and Taneja, {Krishan L.} and Singer, {Robert H.}",
year = "1995",
month = "12",
day = "25",
language = "English (US)",
volume = "23",
pages = "4946--4953",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "24",

}

TY - JOUR

T1 - Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells

AU - Politz, Joan C.

AU - Taneja, Krishan L.

AU - Singer, Robert H.

PY - 1995/12/25

Y1 - 1995/12/25

N2 - Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18 h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.

AB - Cells internalized synthetic oligonucleotides (oligos) in culture. The hybridization of these molecules to target RNA in the living cell was subsequently detected and characterized after fixation of the cells, with or without previous detergent extraction. Hybridized oligo was distinguished from free oligo in the cell using an in situ reverse transcription technique. This assay exploited the ability of the hybridized oligo to prime synthesis of a specific cDNA strand; unhybridized oligo present in the cell could not act as a primer for reverse transcription. Phosphorothioate and fluorochrome-labeled phosphodiester oligo dT were found to enter cells rapidly and hybridize to poly (A) RNA within 30 min. Hybrids containing phosphorothioate oligo dT were detectable in cells after up to 4 h of efflux time. Phosphodiester bonded oligo dT containing covalently-linked fluorochromes appeared more stable in the cell than unmodified phosphodiester oligo dT; hybrids containing these oligos could be detected in cells as long as 18 h after efflux began. The in situ transcription assay was also sensitive enough to detect hybridization of anti-actin oligos to actin mRNA in the cell. It is probable, therefore, that this assay can be used to help assess the efficacy of antisense oligos by their hybridization to a target mRNA in cells or tissues; hybridized oligos are more likely to induce a specific antisense effect. Additionally, this assay will help to identify probes that would be useful as stable hybridization tags to follow RNA movement in living cells.

UR - http://www.scopus.com/inward/record.url?scp=0029564435&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029564435&partnerID=8YFLogxK

M3 - Article

C2 - 8559650

AN - SCOPUS:0029564435

VL - 23

SP - 4946

EP - 4953

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 24

ER -