Design, preparation and testing of suitable probe-receptors for RNA biosensing.
Absolute measurements of a given RNA in a cheap, easy, rapid and reproducible manner using biosensors technology could overcome many of the operative and analytical limits of conventional molecular biology methods. To this end, an integrated approach for the design, synthesis, and connection of RNA probes to the transducing surface of a microgravimetric biosensor has been developed. Suitable probes to be used as the bioreceptors in RNA biosensor were successfully designed by using a purposely developed computational method whose selection criteria are based on the accessibility of target region to probe, on pairing stability of probe-target duplex and on the uniqueness of selected targets over all known expressed sequences from a genome data base. Automated chemical synthesis of selected probes was performed and the oligonucleotides produced were covalently conjugated to the sensing surface of a quartz microbalance. The microgravimetric sensor was tested in a flow chamber by measuring the variation of resonance frequency due to the binding of synthetic target substrates. Specific dose dependent binding was observed. Furthermore, the binding of a transcribed full-length mRNA substrate was successfully monitored under similar conditions.
Tedeschi
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Laboratory of Gene and Molecular Therapy and Laboratory of Sensors and Transducers, Institute of Clinical Physiology, CNR, Area della Ricerca, via Moruzzi,1-56124 Pisa, Italy.
Mercatanti
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Domenici
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Citti
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D001483:Base Sequence; D015374:Biosensing Techniques; D006801:Humans; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D011791:Quartz; D015347:RNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*
D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger; D011791:Quartz; D019853:O(6)-Methylguanine-DNA Methyltransferase