Year of Award:
Molecular & Cellular Analysis Technologies
CLAWSON, GARY A.
Other PI or Project Leader:
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization 'sandwich'. Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (R??) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R??) will initially be detected optically). We have successfully measured R? of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity 'off-chip' synthesis and derivatization of nanowires, R?. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.