Sequential structure probing of cotranscriptional RNA folding intermediates.

Cotranscriptional RNA folding pathways typically involve the sequential formation of folding intermediates. Existing methods for cotranscriptional RNA structure probing map the structure of nascent RNA in the context of a terminally arrested transcription elongation complex. Consequently, the rearrangement of RNA structures as nucleotides are added to the transcript can be inferred but is not assessed directly.

Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide.

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

Systematic analysis of cotranscriptional RNA folding using transcription elongation complex display.

RNA can fold into structures that mediate diverse cellular functions. Understanding how RNA primary sequence directs the formation of functional structures requires methods that can comprehensively assess how changes in an RNA sequence affect its structure and function. Here we have developed a platform for performing high-throughput cotranscriptional RNA biochemical assays, called Transcription Elongation Complex display (TECdisplay).

Observation of coordinated RNA folding events by systematic cotranscriptional RNA structure probing.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates.

Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long and complex transcripts, we developed a procedure for isolating roadblocked E. coli TECs from an in vitro transcription reaction using solid-phase photoreversible immobilization.

Observation of coordinated cotranscriptional RNA folding events.

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates.

A small molecule that targets the processivity factor of molluscum contagiosum virus has therapeutic potential.

Molluscum contagiosum (MC) is an infectious disease that occurs only in humans with a tropism that is narrowly restricted to the outermost epidermal layer of the skin. Molluscum contagiosum virus (MCV) is the causative agent of MC which produces skin lesions that can persist for months to several years. MCV is efficiently transmitted by direct physical contact or by indirect contact with fomites.

Isolation of synchronized E. coli elongation complexes for solid-phase and solution-based in vitro transcription assays.

Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. coli RNAP TECs from an in vitro transcription reaction.

Cotranscriptional RNA Chemical Probing.

Cotranscriptional folding is a fundamental step in RNA biogenesis and the basis for many RNA-mediated gene regulation systems. Understanding how RNA folds as it is synthesized requires experimental methods that can systematically identify intermediate RNA structures that form during transcription. Cotranscriptional RNA chemical probing experiments achieve this by applying high-throughput RNA structure probing to an in vitro transcribed array of cotranscriptionally folded intermediate transcripts.

Purification of synchronized Escherichia coli transcription elongation complexes by reversible immobilization on magnetic beads.

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more nucleoside triphosphates from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes but has not been optimized for RNA-centric assays.

Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking.

Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription "roadblocking" approaches halt transcription elongation using a protein blockade that is non-covalently attached to the template DNA.

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision*.

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution.

Preparation of E. coli RNA polymerase transcription elongation complexes by selective photoelution from magnetic beads.

In vitro studies of transcription frequently require the preparation of defined elongation complexes. Defined transcription elongation complexes (TECs) are typically prepared by constructing an artificial transcription bubble from synthetic oligonucleotides and RNA polymerase. This approach is optimal for diverse applications but is sensitive to nucleic acid length and sequence and is not compatible with systems where promoter-directed initiation or extensive transcription elongation is crucial.

Chemical roadblocking of DNA transcription for nascent RNA display.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current transcription "roadblocking" approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal transcription reaction.

Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor.

Protein disulfide isomerase (PDI, PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies, although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models.

A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control.

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox.

SnapShot: RNA Structure Probing Technologies.

Chemical probing coupled to high-throughput sequencing offers a flexible approach to uncover many aspects of RNA structure relevant to its cellular function. With a wide variety of chemical probes available that each report on different features of RNA molecules, a broad toolkit exists for investigating in vivo and in vitro RNA structure and interactions with other molecules.

High-throughput determination of RNA structures.

RNA performs and regulates a diverse range of cellular processes, with new functional roles being uncovered at a rapid pace. Interest is growing in how these functions are linked to RNA structures that form in the complex cellular environment. A growing suite of technologies that use advances in RNA structural probes, high-throughput sequencing and new computational approaches to interrogate RNA structure at unprecedented throughput are beginning to provide insights into RNA structures at new spatial, temporal and cellular scales.

LX2761, a Sodium/Glucose Cotransporter 1 Inhibitor Restricted to the Intestine, Improves Glycemic Control in Mice.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice.

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks.

Discovery of LX2761, a Sodium-Dependent Glucose Cotransporter 1 (SGLT1) Inhibitor Restricted to the Intestinal Lumen, for the Treatment of Diabetes.

The increasing number of people afflicted with diabetes throughout the world is a major health issue. Inhibitors of the sodium-dependent glucose cotransporters (SGLT) have appeared as viable therapeutics to control blood glucose levels in diabetic patents. Herein we report the discovery of LX2761, a locally acting SGLT1 inhibitor that is highly potent in vitro and delays intestinal glucose absorption in vivo to improve glycemic control.

Cotranscriptional folding of a riboswitch at nucleotide resolution.

RNAs can begin to fold immediately as they emerge from RNA polymerase. During cotranscriptional folding, interactions between nascent RNAs and ligands are able to direct the formation of alternative RNA structures, a feature exploited by noncoding RNAs called riboswitches to make gene-regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function.

RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs.

RNAs assume sophisticated structures that are active in myriad cellular processes. In this review, we highlight newly identified ribozymes, riboswitches, and small RNAs, some of which control the function of cellular metabolic and gene expression networks. We then examine recent developments in genome-wide RNA structure probing technologies that are yielding new insights into the structural landscape of the transcriptome.

A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat.