A modular platform for bioluminescent RNA tracking.

A complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we report a bioluminescent platform for serial imaging of RNAs.

Bioorthogonal cyclopropenones for investigating RNA structure.

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments.

In vitro selection of human cerebrospinal fluid-specific aptamers using clinical samples.

Background: Cerebrospinal fluid (CSF) leaks may occur due to numerous etiologies and are associated with severe morbidity. Currently in the U.S.

Multimodal diagnosis of cerebrospinal fluid rhinorrhea: State of the art review and emerging concepts.

Objective: Currently, diagnosis of cerebrospinal fluid (CSF) rhinorrhea relies on a multimodal approach, increasing costs and ultimately delaying diagnosis. In the United States and internationally, the crux of such a diagnosis relies on confirmation testing (via biomarkers) and localization (e.g.

Inhibition of ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 () gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory.

Inhibition of CPEB3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs, and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element binding protein 3 () gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory.

Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing.

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples.

Self-cleaving ribozymes: substrate specificity and synthetic biology applications.

Various self-cleaving ribozymes appearing in nature catalyze the sequence-specific intramolecular cleavage of RNA and can be engineered to catalyze cleavage of appropriate substrates in an intermolecular fashion, thus acting as true catalysts. The mechanisms of the small, self-cleaving ribozymes have been extensively studied and reviewed previously. Self-cleaving ribozymes can possess high catalytic activity and high substrate specificity; however, substrate specificity is also engineerable within the constraints of the ribozyme structure.

Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate*.

A mechanism of nucleoside triphosphorylation would have been critical in an evolving "RNA world" to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. Previous reports have demonstrated that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under prebiotically-relevant conditions, but their reaction rates were unknown and the influence of reaction conditions not well-characterized.

Single-pass transcription by T7 RNA polymerase.

RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system.

Co-transcriptional Analysis of Self-Cleaving Ribozymes and Their Ligand Dependence.

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors.

Regulation of mRNA translation by a photoriboswitch.

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the isoform of a stiff-stilbene (amino-SS)-a rapidly and reversibly photoisomerizing small molecule.

Large Phenotypic Enhancement of Structured Random RNA Pools.

Laboratory evolution of functional RNAs has applications in many areas of chemical and synthetic biology. In vitro selections critically depend on the presence of functional molecules, such as aptamers and ribozymes, in the starting sequence pools. For selection of novel functions the pools are typically transcribed from random-sequence DNA templates, yielding a highly diverse set of RNAs that contain a multitude of folds and biochemical activities.

High-throughput methods in aptamer discovery and analysis.

Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences.

Kinetic Parameters of trans Scission by Extended HDV-like Ribozymes and the Prospect for the Discovery of Genomic trans-Cleaving RNAs.

Hepatitis delta virus (HDV)-like ribozymes are self-cleaving catalytic RNAs with a widespread distribution in nature and biological roles ranging from self-scission during rolling-circle replication in viroids to co-transcriptional processing of eukaryotic retrotransposons, among others. The ribozymes fold into a double pseudoknot with a common catalytic core motif and highly variable peripheral domains. Like other self-cleaving ribozymes, HDV-like ribozymes can be converted into trans-acting catalytic RNAs by bisecting the self-cleaving variants at non-essential loops.

DNA synthesis from diphosphate substrates by DNA polymerases.

The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the s of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively.

Allosteric Modulation of the Faecalibacterium prausnitzii Hepatitis Delta Virus-like Ribozyme by Glucosamine 6-Phosphate: The Substrate of the Adjacent Gene Product.

Self-cleaving ribozymes were discovered 30 years ago and have been found throughout nature, from bacteria to animals, but little is known about their biological functions and regulation, particularly how cofactors and metabolites alter their activity. A hepatitis delta virus-like self-cleaving ribozyme maps upstream of a phosphoglucosamine mutase (glmM) open reading frame in the genome of the human gut bacterium Faecalibacterium prausnitzii. The presence of a ribozyme in the untranslated region of glmM suggests a regulation mechanism of gene expression.

A Microwell-Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging.

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements.

Multiplex Aptamer Discovery through Apta-Seq and Its Application to ATP Aptamers Derived from Human-Genomic SELEX.

Laboratory-evolved RNAs bind a wide variety of targets and serve highly diverse functions, including as diagnostic and therapeutic aptamers. The majority of aptamers have been identified using in vitro selection (SELEX), a molecular evolution technique based on selecting target-binding RNAs from highly diverse pools through serial rounds of enrichment and amplification. In vitro selection typically yields multiple distinct motifs of highly variable abundance and target-binding affinities.

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes.

Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity - a "pinch" that results in rate acceleration by almost three orders of magnitude.

RNA motif search with data-driven element ordering.

Background: In this paper, we study the problem of RNA motif search in long genomic sequences. This approach uses a combination of sequence and structure constraints to uncover new distant homologs of known functional RNAs. The problem is NP-hard and is traditionally solved by backtracking algorithms.

Improving the odds: Influence of starting pools on in vitro selection outcomes.

As with any outcome of an evolutionary process, the success of in vitro selection experiments depends critically on the starting population. In vitro selections isolate functional nucleic acids that fold into specific structures and form unique binding and catalytic sites. The selection outcomes therefore depend on the molecular and structural diversity of the initial pools.