The detection of RNA provides for rapid identification of microorganisms, gene regulation analysis, molecular diagnostics, and detection of viral persistency. However, there is still a need for RNA detection methods that are rapid, quantitative, sensitive, and amenable for direct monitoring in a variety of matrices. The work presented within describes a method that fulfills these characteristics, and will be ideal for the detection of viral persistency, a scenario in which viruses hide within a specific cell type while remaining few in number. Stem-loop probes (SLPs) are a class of nucleic acid biosensors that are single-stranded oligonucleotide probes containing a loop region (about 15-30 nt) complementary to a target sequence. This loop region is flanked by two short self-complementary regions (about 5-7 nt) known as the stem, which are typically conjugated to a fluorophore and a quencher. The SLP switches from closed to open conformations upon hybridization of a complementary target to the loop region. In the closed conformation, fluorescence is quenched by an energy transfer mechanism, whereas in the open conformation this quenching effect is removed, and fluorescence can be measured. The inherent signal transduction mechanism of these probes yields superior specificity and the ability to perform detection in real time without separation of unhybridized probes. However, currently available SLPs have limited sensitivity. Addressing these problems present in current SLPs and generating effective solutions will allow SLPs to serve as a platform of low-level RNA detection, as is necessary in the case of viral persistency. Toward that end, we hypothesize that the development of a bioluminescent SLP (BSLP) would enhance sensitivity of current SLP-based detection systems by retaining the rugged versatility and broad applicability of its fluorescent counterparts while exhibiting low background and high detection sensitivity. Our hypothesis will be tested by pursuing three specific aims, 1) Enhance analytical performance of BSLPs and develop facile BSLP synthesis methods, 2) Incorporate bioluminescent enzymes possessing high substrate turnover as well as thermostable, long-lived, highly active photo proteins into BSLP design, 3) Design, develop and optimize BSLPs for the detection of viral persistency using HIV persistency as a model system in cell and blood samples. The proposed work is innovative because it introduces a novel, new sensing strategy that seamlessly combines the high sensitivity of bioluminescent proteins and the specificity of SLPs. This provides a means for the rapid detection of low levels of viral RNA in any sample matrix such as would exist in the case of viral persistency, solving a problem current technologies aren't capable of addressing. This research is significant because it is expected to provide a highly sensitive tool for the detection of nucleic acids that will allow for the detection of viral persistency. This, in turn, wll have a significant impact on infectious disease diagnostics and therapeutic monitoring.
Viral persistency is a critical issue in the areas of both analytical research as well as clinical application due to the fact that certain viruses possess th ability to hide in a specific cell type, while maintaining low levels of characteristic DNA/RNA. Hence, new RNA detection methods that are highly sensitive, rapid, and which provide for the direct detection of these molecules are needed. In that regard, the goal of the proposed work is to develop new bioluminescent stem-loop probes (BSLPs) that provide unprecedented sensitivity and selectivity for RNA sensing in a rapid manner in any sample matrix.
