Riboswitch Screening Enabled By Microfluidic Electrophoretic Mobility Shift Assays (EMSAs)

Riboswitches are RNA cis-regulatory genetic control elements in prokaryotic cells. The molecular conformation of riboswitch encodes important information regarding its metabolic function and is closely tied to ligand binding in the cells. Upon binding, riboswitch conformation changes, in turn controlling gene expression at the transcriptional level. In exciting new research directions, riboswitch binding interactions have been targets for antibacterial drug development. A key aspect of screening involves compound library assessment to identify effective binders for riboswitches and subsequent binding affinity characterization.

Electrophoretic mobility shift assays (EMSAs) are the most common approach for interaction screening studies. Generally, the electrophoretic mobility depends on the size and charge of a molecule and EMSAs report analyte mobility shifts as species migrate through the nanoporous sieving medium, in turn enabling probing of molecular binding, reflecting the conformation induced molecular size change. However, conventional slab-gel polyacrylamide electrophoresis (Slab-PAGE) based EMSAs struggle in quantifying conformation based small shifts in mobility due to long durations required to electrophoretically resolve these species. In addition, the standard slab-gel format is not amenable for scale-up, which hinders the development for high-throughput screening. To fill this technical gap, we build on our recently developed multiplexed free-standing polyacrylamide gel (fsPAG) microsystem [1] to introduce quantitative, robust and rapid EMSAs for assessment of small riboswitch conformation change, with up to 500-fold increase in throughput.

In this as of yet not reported study, we investigate key factors contributing to separation performance in the new fsPAG format and utilize engineering approaches to optimize for a reproducible and quantitative EMSA. In particular, we detail (1) PAG structural non-uniformity resulting from UV photopatterning and (2) moisture evaporation due to the open gel environment. We then demonstrate the EMSA on a single-plex fsPAG with cyclic-di-GMP riboswitch. EMSA on fsPAG format provides substantially higher throughput than slab-PAGE.  We observe a 200 fold reduction in required separation time and 30 fold reduction in separation distance. A subsequent EMSA on a 96-plex fsPAG provides quantitative measurement of the binding affinity of the riboswitch binding pair. The ability for fsPAG EMSAs to offer tight control of conditions enables statistical testing of observed shifts. The EMSA fsPAG device offers a multiplexed format with promise for high-throughput binding screening for a range of applications.

Reference:

[1] Duncombe, T. A., Herr, A. E., Lab Chip 2013, 13, 2115-2123.