The central theme of this proposal is to develop and apply molecular beacon based biosensing arid bioimaging technologies for quantitative mRNA determination in living specimen. The ability to detect and quantitate changes in gene expression, especially in real-time and with a degree of sensitivity suitable to monitor minor changes at a single cell level, will have considerable value in disease diagnosis, disease mechanism research and drug discovery. It is also necessary to be able to distinguish between expression products of genes that are related since it is becoming increasingly clear that single nucleotide changes can (I) have significant effects on the biological activity of a gene product and/or (ii) affect the response to a drug molecule. While revolutionary advances in high throughput gene sequencing now allow us to detect single base alterations at the genetic level, similar bioanalytical methods to detect gene products in living cells are yet to be developed. We will use a recently developed DNA probe, molecular beacon (MB), for the development of real-time biosensing and bioimaging techniques for ultrasensitive monitoring of DNA/RNA in living specimen. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids. MBs have extremely high selectivity arid excellent analytical sensitivity. MBs can be designed to have (i) selectivity with single base mismatch identification capability, (ii) detection without separation, (iii) sensitivity to detect rare messages and (iv) no cytotoxic effects in order to allow continued monitoring of physiological changes in live cells. They hold great promise for studies in genetics, molecular interactions, and for applications in disease diagnostics, gene therapy and in new drug development. We will concentrate on developing MBs' bioanalytical application as ultrasensitive probes and biosensors for the quantitation of gene products inside living cells and from cellular extracts and tissue samples. The proposed research will capitalize on our extensive research results using MBs as well as our recent advancement in nanotechnology to create ultrasmall biochemical sensors and nanomaterials, in optical imaging at the single molecule level and in single living cell studies. Specifically, we will pursue the following aims in this proposal:
Specific Aim 1 : Development and application of ultrasensitive MB biosensors for mRNA inside living specimen and mRNA products of specific genes implicated in oncogenic transformation.
Specific Aim 2 : Exploration of quantitative applications of MBs inside single living cells for real-time determination of mRNA from genes implicated in cellular metabolism or apoptosis and genes delivered in gene therapy. Our overall strategy is to start parallel experiments in MB biosensor development/application and in real-time mRNA quantitative monitoring. The biosensor development will mainly emphasize purity of MB, new design for surface immobilization, miniaturizatIon, better understanding of the surface bound MBs in hybridization, better application of the MB sensors in a variety of cellular extract samples and finally living specimen samples. Real-time monitoring of mRNA will be dealt with mainly in the following areas: First, single molecule imaging techniques adopted for living specimen monitoring with ultrasensitivity; Second, MB design and better synthesis for ultrasensitive monitoring and fast hybridization kinetics; Third, quantitative methodology for living cell studies; Fourth, real-time determination of mRNA from genes implicated in cancer cells and genes delivered in gene therapy. Our project is an integrated one in which ultrasensitive biosensors and imaging techniques will be developed; MB design and synthesis will be improved; ratiometric quantitation method will be utilized for mRNA determination; new insights into mRNA's role in cellular function and in cellular metabolism or apoptosis will be obtained. By carrying out this research plan, we will have developed better and more efficient DNA/RNA biosensors, studied hybridization processes at the biosensor surface. Ve will have improved MB design, synthesis and usage. We will have developed quantitative tools for real-time monitoring of mRNA with ultrasensitivity. We will have acquired quantitative evidences of gene expression and replication inside living cells. We fully anticipate the research results from this proposal will have a major impact on a few important areas such as disease diagnosis, disease mechanism studies, gene therapy and drug development.
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