Sequencing microRNAs, with single-base discrimination, using a nanopore device and an Osmium tag.

PROJECT SUMMARY The goal of this work is to develop an inexpensive, ultra-fast, minimally-invasive medical test to sequence and quantitate small RNAs from biological fluids. Small RNAs, shorter than 200 nucleotides (nt) comprise several groups of non-coding RNAs with preserved regulatory functions. Small RNAs are abundant and surprisingly stable in blood/urine. Current literature is flooded with studies identifying such small RNAs as reliable biomarkers for most diseases. These studies suggest that a comprehensive small RNA panel will reflect the health/aging status of an individual, response to stress, changes in medication, onset and/or progress of disease. Current small RNA profiling assays are expensive, involve extensive infrastructure, and employ time- consuming and error-prone sample preparation. These assays also miss or misidentify post-transcriptionally modified bases (PTM) which are known to influence RNA stability, specificity, and function. On the contrary, nanopore-based technologies promise inexpensive portable devices, direct RNA profiling including identification of PTM bases, and fast assay turn-around. Still the only commercially available nanopore-based device(s) from Oxford Nanopore Technologies (ONT) are not qualified to sequence RNAs shorter than 200 bases. Such limitation prevents sequencing of most, if not all, non-coding RNAs found in biological fluids, and limits the usability of nanopore technology in minimally-invasive medical diagnostic assays. We will use commercially available nanopore devices and optimize the testing conditions in order to profile small RNAs in biological fluids. Yenos Analytical LLC is singled out for such effort, as we have developed proprietary technology to selectively tag RNAs with a bulky Osmium label. The bulky label slows down the voltage-driven translocation of these RNA surrogates via a nanopore, and yields a readable ion current vs time (i-t) signal that serves as the fingerprint of the specific RNA molecule. Recent work funded by a Phase I SBIR NHGRI grant has also shown that some of the slowest translocations of these tagged RNAs using the ONT device(s) embody sequencing information. In order to increase the number of the slow events, we will exploit non-covalent probes that bind RNA. This binding will reduce the effective negative charge of the RNA molecule, and slowly release it under the influence of the voltage drop in proximity to the nanopore’s entry. As non-covalent probes, we will evaluate cationic polyamines such as spermidine and spermine, cationic peptides, and RNA binding proteins. The combination of nucleic acid selective labeling technology with nanopore-device based detection is novel, and single-handedly carried out by Yenos Analytical LLC. We propose to optimize this combination of technologies towards profiling small RNAs from biological fluids. Such a medical assay will directly impact medical testing, prevention, diagnosis, and cure of disease, materialize the promise of personalized medicine, reduce the ballooning cost of health care, and enable diagnostic medical tests in remote locations with minimal infrastructure.PROJECT NARRATIVE: An explosion in recent studies solidifies the importance of small RNAs in biological fluids as disease biomarkers. Advances in personalized medicine for diagnosis and treatment of disease require minimally-invasive, inexpensive, ultra-fast medical tests. Nanopore-based devices are commercially available, portable and inexpensive. If contrived to provide high accuracy in identification and quantitation of small RNAs from biological fluids, as described in this proposal, these nanopore-based devices will revolutionize personalized medicine and reduce the cost of health care.