Development of Automated Infrastructures in Microfluidics for Site-Specific Labeling of RNA (PLOR)

Position‐specific labeling of RNA (PLOR) is a technique that enables incorporation of modified nucleotides at defined positions within an RNA transcript. However, traditional PLOR workflows rely on manual sequential reagent delivery, leading to significant time consumption and reagent waste when labeling large RNA strands. In this study, we developed an automated, multiplexed pneumatic peristaltic pumping platform integrated into a multilayer PDMS microfluidic chip that enables precise, low‐dead‐volume reagent delivery for PLOR. We first studied how membrane thickness, valve geometry, and channel dimensions influence single‐channel peristaltic flow and optimized a metering module that achieves a volumetric flow rate of approximately 370 nL/min at 1.67 Hz. We then conducted simulated reagent routing using colored dyes to evaluate an eight‐channel multiplexing architecture—demonstrating how combinations of reagents could be driven by the pneumatic peristaltic pumps and routed through multiplexed valves into designated channels. During RNA transcription and specific labeling these streams would include different NTP mixes, modified nucleotides, and buffer solutions. In parallel, we implemented an on‐chip PLOR platform in which reagents are driven by constant‐pressure pumps and delivered into reaction chambers via pneumatic valves. This system is capable of PLOR on any DNA template by pausing transcription at defined positions and incorporating user‐selected modified nucleotides. In this study, 5‐aminoallyl‐UTP was incorporated and subsequently fluorescently tagged, as demonstrated by pausing a custom 69‐nt RNA at positions 11 and 12 for site‐specific labeling. By eliminating manual pipetting, reducing dead volume, and enabling high‐throughput operation, our platform provides a robust, scalable solution for reproducible RNA synthesis and labeling, paving the way for integrated biosensing, single‐molecule fluorescence, and advanced structural studies.