SNP detection in mRNA in living cells using allele specific FRET probes

PLoS One. 2013 Sep 9;8(9):e72389. doi: 10.1371/journal.pone.0072389. eCollection 2013.

Abstract

Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2'OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Base Sequence
  • Cell Line, Tumor
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemistry
  • Genes, ras
  • Genotyping Techniques*
  • Mice
  • Mice, Inbred BALB C
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Phosphorothioate Oligonucleotides / chemistry
  • Phosphorothioate Oligonucleotides / genetics
  • Point Mutation
  • Polymorphism, Single Nucleotide*
  • RNA Probes / chemistry
  • RNA Probes / genetics
  • RNA Stability
  • RNA, Messenger / genetics*
  • Sensitivity and Specificity
  • Single-Cell Analysis

Substances

  • Fluorescent Dyes
  • Oligonucleotides
  • Phosphorothioate Oligonucleotides
  • RNA Probes
  • RNA, Messenger
  • locked nucleic acid

Grants and funding

This work was supported in part by the grants from the Lewis Family Trust, the Israeli Centre of Research Excellence (I-CORE), Gene Regulation in Complex Human Disease, Center No 41/11; FTA: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund awarded to DP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.