Poster abstracts
Poster number 16 submitted by Rachel Hemmerlin
RNA engineering to map the folding pathway of a catalytic RNA using optical tweezers and to explore the promise of mass spectrometry-based cancer diagnostics
Rachel M. Hemmerlin (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Ehsan Akbari (Department of Physics, The Ohio State University, Columbus, OH 43210), Henry C. Arthur, Anju Sheregar, Ayesha Seth (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Abraham Badu-Tawiah (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Michael G. Poirier (Department of Physics, The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210)
Abstract:
Ribonuclease P (RNase P), a catalytic ribonucleoprotein (RNP), is responsible for the 5' maturation of precursor tRNAs.^1,2 The RNP form of the enzyme is composed of a catalytic RNase P RNA (RPR) and one or more RNase P proteins (RPPs).^3 Archaeal RNase P (RPR + 5RPP) has an intermediate subunit complexity compared to its bacterial (RPR + 1RPP) and eukaryotic (RPR + ≤10RPPs) cousins. The archaeal version is an ideal surrogate for the intractable eukaryotic relative. All RPRs contain a catalytic domain and a specificity domain. We previously used ensemble FRET to show that an archaeal RPR undergoes Mg2+-induced structural remodeling, albeit through different paths, in the presence or absence of RPPs.^4 The effect(s) of individual RPPs on the RPR structure is unknown. Here, we explore the use of optical tweezers to unravel the unfolding and folding pathways of an archaeal RPR at different [Mg2+]. Our model is the RNase P from Methanobrevibacter smithii (Msm), a mesophilic archaeon. For our force-spectroscopy studies, we first prepared a Msm RPR with both 5' and 3' single-stranded extensions to enable annealing to DNA tethers designed to have overhangs complementary to the engineered extensions. Force-extension curves show that Mg2+ stabilizes the RPR structure, establishing the feasibility of these single-molecule experiments to yield insights into RPR structural changes essential for RNase P assembly and catalysis.
The detection of cancer biomarkers in blood offers new prospects for diagnostics. These markers include circulating tumor nucleic acids (ctNAs) comprising of cell-free DNA/RNA released from tumor cells.^5 Many messenger RNAs and long noncoding RNAs^6,7 are upregulated in the blood of cancer patients highlighting their potential to be used in non-invasive cancer diagnosis.^8 Here, we seek to develop a method to detect specific RNAs by mass spectrometry (MS). For in vitro proof-of-concept, we leveraged the Msm RPR with 5' and 3' extensions already in hand (above). Streptavidin beads and biotinylated DNA were used to capture the Msm RPR for subsequent interrogation with a second DNA that contains a cleavable ionic probe for MS detection. These studies will provide a foundation for developing a paper-based, cheap cancer diagnostic method.
References:
(1) Altman, S. (2007) Mol. BioSyst. 3 (9), 604–607.
(2) Mondragón, A. (2013) Annu. Rev. Biophys. 42, 537–557.
(3) Phan et al. (2021) Trends Biochem. Sci. 46 (12), 976–991.
(4) Marathe et al. (2021) Nucleic Acids Res. 49 (16), 9444–9458.
(5) Stejskal et al. (2023) Mol. Cancer 22, 15.
(6) He et al. (2021) Front. Oncol. 11, 632834.
(7) Badowski et al. (2022) NPJ Precis. Onc. 6 (1), 1–18.
(8) Wang et al. (2021) Front. Med. 8.
Keywords: RNase P, single-molecule force measurements, cancer diagnosis