BPS2026 – Pore-based RNA evaluation for control of integrity, sequence, and errors—Quality control (PRECISE-QC)

Solid-phase synthesis is currently the gold standard for synthesizing short RNA oligos (<50 nt). Their single base incorporation cycle allows for site specific inclusion of RNA modifications that are beneficial for many RNA therapeutic applications. However, varying factors such as reagent quality, humidity, and oligo sequence can commonly lower the coupling efficiency leading to poor yields and low sequence fidelity. This is especially troublesome when attempting to synthesize long RNA oligos (>50 nt). Furthermore, current quality control methods such as gel electrophoresis and HPLC can only provide insight toward RNA length distribution. While mass spectrometry can provide composition information, it is limited to short oligos. Here, we have developed a quality control metric using Oxford Nanopore Technology’s direct RNA sequencing (ONT DRS) tools. Our pipeline was used to assess and refine production of solid-phase synthesized 101 nt single-guide RNAs (sgRNAs) by providing insight on truncated byproducts, sequence fidelity, reagent quality, and modification detection (m 6 A, Ψ, Nms). We believe our method can serve as an integral RNA synthesis quality control pipeline that will ensure the integrity and accuracy of RNA products.