title: SNP detection in mRNA in living cells using allele specific FRET probes.
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.
authors.affiliation: Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel ; Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Liya
authors.initials: L
authors.lastname: Dahan
authors.forename: Lingyan
authors.initials: L
authors.lastname: Huang
authors.forename: Ranit
authors.initials: R
authors.lastname: Kedmi
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Dan
authors.initials: D
authors.lastname: Peer
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D011905:Genes, ras; D060005:Genotyping Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D015347:RNA Probes / Q000737:chemistry / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics*; D012680:Sensitivity and Specificity; D059010:Single-Cell Analysis
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D054735:Phosphorothioate Oligonucleotides; D015347:RNA Probes; D012333:RNA, Messenger; C477371:locked nucleic acid
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