Novel Nanoprobes to Detect mRNA in situ Directed Against Mouse Pyruvate Dehydrogenas

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Novel nanoscale synthetic locked nucleic acid (LNA) probes incorporating molecular beacon (MB) technology were developed. These novel MBs contained 2’-N-(pyren-1-yl)carbonyl-2’-amino-LNA monomers incorporated in the loop region. Specificity and sensitivity of MB designed to target the mRNA of mouse pyruvate dehydrogenase complex, component X (Pdhx) mRNA were tested. Using antisense and sense RNA strands of mouse Pdhx synthesized by in vitro transcription, binding specificity of MBs was examined. Incubation of antisense mRNA with the MB showed a dose-response relationship and linear increase. Cellular uptake of MBs by mouse 3T3-L1 cells and in-cell fluorescence of MB-target mRNA complexes was demonstrated in a dose dependent manner. A linear increase in fluorescence emitted from the cells was observed with the increasing amount of transfected MB. In addition, we examined the stability of the MB fluorescence signal after MB were taken up by the cells. We observed that fluorescence emission was stable even at the maximum time-point tested (48 h). Finally, to test the effect of the backbone structure of MB on the resulting fluorescent signal intensity, comparisons were performed between MB having phosphorothioate (PS-DNA) and 2’ O-Me RNA as backbone elements. We demonstrated that MB with 2’ O-Me RNA backbone provide enhanced fluorescence, compared to MB containing PS-DNA backbone.

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Journal: TechConnect Briefs
Volume: 3, Nanotechnology 2010: Bio Sensors, Instruments, Medical, Environment and Energy
Published: June 21, 2010
Pages: 300 - 303
Industry sector: Medical & Biotech
Topics: Biomaterials, Materials for Drug & Gene Delivery
ISBN: 978-1-4398-3415-2