Live cell screening to identify RNA-binding small molecule inhibitors of the pre-let-7-Lin28 RNA-protein interaction.

Dysregulation of the networking of RNA-binding proteins (RBPs) and RNAs drives many human diseases, including cancers, and the targeting of RNA-protein interactions (RPIs) has emerged as an exciting area of RNA-targeted drug discovery. Accordingly, methods that enable the discovery of cell-active small molecule modulators of RPIs are needed to propel this emerging field forward. Herein, we describe the application of live-cell assay technology, RNA interaction with protein-mediated complementation assay (RiPCA), for high-throughput screening to identify small molecule inhibitors of the pre-let-7d-Lin28A RPI. Utilizing a combination of RNA-biased small molecules and virtual screening hits, we discovered an RNA-binding small molecule that can disrupt the pre-let-7-Lin28 interaction demonstrating the potential of RiPCA for advancing RPI-targeted drug discovery.

Optimization of RiPCA for the Live-Cell Detection of Pre-MicroRNA-Protein Interactions.

Advancements in methods for identifying RNA-protein interactions (RPIs) on a large scale has necessitated the development of assays for validation of these interactions, particularly in living cells. We previously reported the development of RiPCA (RNA interaction with protein-mediated complementation assay) to enable the cellular detection of the well-characterized interaction between the pre-microRNA, pre-let-7, and its RNA-binding protein (RBP) partner Lin28. In this study, the applicability of RiPCA for the detection of putative pre-miRNA-protein interactions was explored using an improved RiPCA protocol, termed RiPCA 2.0. RiPCA 2.0 was adapted to detect the sequence specificity of the RBPs hnRNP A1, Msi1, and Msi2 for reported pre-microRNA binding partners. Additionally, the ability of RiPCA 2.0 to detect site-specific binding was explored. Collectively, this work highlights the versatility of RiPCA 2.0 in detecting cellular RPIs.

RiPCA: An Assay for the Detection of RNA-Protein Interactions in Live Cells.

Increasing interest in studying and modulating the interactions between RNAs and their RNA-binding proteins has indicated the need for enabling technologies. Existing means of detecting RNA-protein interactions (RPIs) are often limited to biochemical or post-lysis methods or cell-based methods that require the addition of an RNA-based affinity tag, such as the MS2 hairpin, precluding them from use in detecting small or highly processed RNAs. Taking advantage of bioorthogonal chemistry- and split-luciferase-based technologies, we developed an assay for the detection of RPIs in live cells. This article details the protocol and design considerations for RiPCA, or RNA interaction with Protein-mediated Complementation Assay. © 2022 Wiley Periodicals LLC.

A live-cell assay for the detection of pre-microRNA-protein interactions.

Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined that this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.

Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.