Browsing by Subject "Circulating RNA"
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Item Thermostable group II intron reverse transcriptases and their applications in next generation RNA sequencing, diagnostics, and precision medicine(2016-05) Qin, Yidan; Lambowitz, Alan; Iyer, Vishwanath R; Krug, Robert M; Russell, Rick; Stevens, Scott W; Sullivan, Christopher SThermostable group II intron reverse transcriptases (TGIRTs) from thermophilic bacteria are advantageous for biotechnological applications that require cDNA synthesis, such as RT-qPCR and RNA-seq. TGIRTs have higher thermostability, processivity and fidelity than conventional retroviral RTs, along with a novel end-to-end template-switching activity that attaches RNA-seq adapters to target RNAs without RNA ligation. First, I optimized the TGIRT template-switching method for RNA-seq analysis of small non-coding RNAs (ncRNAs). I showed that TGIRT-seq gives full-length reads of tRNAs, which are refractory to retroviral RTs, and enables identification of a variety of base modifications in tRNAs by distinctive patterns of misincorporated nucleotides. With collaborators, I developed an efficient and quantitative high-throughput tRNA sequencing method, identified RNAs bound by the human interferon-induced protein IFIT5, yielding new insights into its functions in tRNA quality control and innate immunity, and uncovered a novel mRNA-independent mechanism for elongation of nascent peptides. Second, I developed a new, streamlined TGIRT-seq method for comprehensive analysis of all RNA size classes in a single RNA-seq. This method enables RNA-seq library construction from <1 ng of fragmented RNAs in <5 h. By using the method, I showed that human plasma contains large numbers of protein-coding and long ncRNAs together with diverse classes of small ncRNAs, which are mostly present as full-length transcripts. With collaborators, I showed that TGIRT-seq analysis of circulating RNAs identified potential biomarkers at different stages of multiple myeloma and may provide a sensitive, non-invasive diagnostic tool for a variety of human diseases. Finally, I adapted TGIRTs for use in mapping of RNA structures and RNA-protein interaction sites, and identification of RNA targets of cellular RNA-binding proteins. My research led to a series of new biological insights, which would have been difficult or impossible to obtain by current methods, and established TGIRTs as a tool for a broad range of applications in RNA research and diagnostics.