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.
Highlights
Much effort has been devoted in the past decade to developing nanostructured molecular probes for RNA live cell imaging [1]
Previous work have shown that the optimal distance varied with Fluorescence Resonance Energy Transfer (FRET) pairs and that FRET pairs with closer emission/excitation wavelengths did better farther apart and those with spectra far apart did better when positional spatially closer
It could serve as a powerful tool for detection and diagnostics and will significantly impact drug discovery and medical diagnostics including shortening the duration of genetic tests procedures
Summary
Much effort has been devoted in the past decade to developing nanostructured molecular probes for RNA live cell imaging [1]. In the field of cancer diagnostics and targeted delivery, most research today is aimed at finding extracellular protein markers (Tumor Associated Antigens, TAA) exclusively or differently expressed in tumors. Discovery of new TAAs is extremely challenging and time consuming whereas tumor genetic alterations (TGA) could be applied by genomic sequencing. Designing a practical method for monitoring genetic alterations in living cells could be used as a simple method for detection of transformed cells. The main challenge of live genetic detection is the ability to produce a high signal to background ratio with high specificity since unbound probe cannot be removed by stringent washing, as is normally employed in nucleic acid hybridization based assays
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