Problem
RNA molecules that are newly transcribed from the corresponding DNA sequence contain intron and exon sequences. Introns are removed by a process called RNA splicing, where the introns are removed and the exons are ligated to form the mature messenger RNA (mRNA) and full-length excised intron RNAs (FLEXIRs). The majority of FLEXIRs and the genes encoding them are either cell- or tissue-type specific, reflecting differences in gene expression, alternative splicing, and differential stability of the FLEXIR RNAs. In addition to FLEXIRs, laboratory experiments have identified fragments of excised intron RNAs as another category of potential biomarkers. Sequencing of the excised intronic RNA can identify disease states and inform treatment.
Solution
A method using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) has been developed to identify thousands of short, full-length excised intron RNAs (FLEXIRs) in unfragmented RNA preparations from human cells, tissues, and plasma. Initial evaluations of FLEXIRs and the genes encoding them indicated hundreds to thousands of easy-to-detect differences in matched healthy and breast cancer tissues from two patients with different breast cancer subtypes and a human breast cancer cell line. Since many FLEXIRs are highly structured RNAs, their initial detection and characterization are performed optimally by use of TGIRT-seq. This technique can provide end-to-end sequence reads of the structured RNAs.
The inventors envision the use of the TGIRT-seq technique to identify optimal combinations of FLEXIR and FLEXIR-encoding-gene disease biomarkers which can be incorporated into targeted RNA panels through the application of different types of readouts such as RT-qPCR, microarrays, hybridization-based assay techniques, and targeted RNA-seq. These RNA panels show potential for application in the diagnosis and routine monitoring of disease progression and response for the treatment of nearly all diseases, and in monitoring applications such as response to toxic chemicals or radiation exposure.
About the inventor
Dr. Alan Lambowitz is the Mr. and Mrs. A. Frank Smith, Jr. Regents Chair in Molecular Biology at The University of Texas at Austin and a Professor of Oncology in the Department of Oncology at the Dell Medical School. Dr. Lambowitz’s primary research interests are in microbiology, immunology, infectious diseases, molecular biology, and genetics. He has authored hundreds of technical publications over the past fifty years and has been the recipient of numerous awards. The focus of his work has broad applicability in the analysis of human cellular, exosomal, and plasma RNAs for RNA diagnostics and the liquid biopsy of human diseases, as well as in biomarker identification.
