Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural Principles of Fluorescent RNA Aptamers.

ABSTRACT: Several aptamer RNAs have been selected in vitro that bind to otherwise weakly fluorescent small molecules and enhance their fluorescence several thousand-fold. By genetically tagging cellular RNAs of interest with these aptamers and soaking cells in their cell-permeable cognate small-molecule fluorophores, it is possible to use them to study RNA localization and trafficking. These aptamers have also been fused to metabolite-binding RNAs to generate fluorescent biosensors. The 3D structures of three unrelated fluorogenic RNAs have been determined, and reveal a shared reliance on base quadruples (tetrads) to constrain the photo-excited chromophore. The structural diversity of fluorogenic RNAs and the chemical diversity of potential fluorophores to be activated are likely to yield a variety of future fluorogenic RNA tags that are optimized for different applications in RNA imaging and in the design of fluorescent RNA biosensors.

SUBMITTER: Trachman RJ

PROVIDER: S-EPMC7699827 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structural Principles of Fluorescent RNA Aptamers.

Trachman Robert J RJ Truong Lynda L Ferré-D'Amaré Adrian R AR

Trends in pharmacological sciences 20170717 10

Several aptamer RNAs have been selected in vitro that bind to otherwise weakly fluorescent small molecules and enhance their fluorescence several thousand-fold. By genetically tagging cellular RNAs of interest with these aptamers and soaking cells in their cell-permeable cognate small-molecule fluorophores, it is possible to use them to study RNA localization and trafficking. These aptamers have also been fused to metabolite-binding RNAs to generate fluorescent biosensors. The 3D structures of t ...[more]

PMID: 28728963

Similar Datasets

Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes.

Project description:RNA-modifying enzymes are difficult to assay due to the absence of fluorometric substrates. Here we show that the Broccoli, a previously reported fluorescent RNA-dye complex, can be modified to contain N(6)-methyladenosine, a prevalent mRNA base modification. Methylated Broccoli is nonfluorescent but, upon demethylation by the RNA demethylases fat mass and obesity-associated protein (FTO) or ALKBH5, it binds and activates the fluorescence of its cognate fluorophore. We describe a high-throughput screen (HTS) for FTO inhibitors using the fluorogenic methylated Broccoli substrate HTS assay, which performs robustly with a Z' factor >0.8 in the LOPAC1280 library. This allowed the identification of novel high-affinity FTO inhibitors. Several of these compounds were selective for FTO over the related demethylase, ALKBH5, and increase methylation of endogenous FTO target mRNAs in cells. Lastly, we show that Broccoli can be modified to contain other base modifications, suggesting that this approach could be generally applicable for assaying diverse RNA-modifying enzymes.

| S-EPMC4800036 | biostudies-literature

Structural computational modeling of RNA aptamers.

Project description:RNA aptamers represent an emerging class of biologics that can be easily adapted for personalized and precision medicine. Several therapeutic aptamers with desirable binding and functional properties have been developed and evaluated in preclinical studies over the past 25years. However, for the majority of these aptamers, their clinical potential has yet to be realized. A significant hurdle to the clinical adoption of this novel class of biologicals is the limited information on their secondary and tertiary structure. Knowledge of the RNA's structure would greatly facilitate and expedite the post-selection optimization steps required for translation, including truncation (to reduce costs of manufacturing), chemical modification (to enhance stability and improve safety) and chemical conjugation (to improve drug properties for combinatorial therapy). Here we describe a structural computational modeling methodology that when coupled to a standard functional assay, can be used to determine key sequence and structural motifs of an RNA aptamer. We applied this methodology to enable the truncation of an aptamer to prostate specific membrane antigen (PSMA) with great potential for targeted therapy that had failed previous truncation attempts. This methodology can be easily applied to optimize other aptamers with therapeutic potential.

| S-EPMC4921272 | biostudies-literature

RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers.

Project description:The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light-up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell-permeable small-molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP-tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA-RNA and RNA-protein interactions using split-FLAP and Förster resonance energy transfer (FRET) systems is given.

| S-EPMC6391945 | biostudies-literature

Structural Principles of CRISPR RNA Processing.

Project description:The Cas6 superfamily, the Cas5d subclass, and the host RNase III endoribonucleases are responsible for producing small RNAs (crRNA) that function in the CRISPR-Cas immunity. The three enzymes may also interact with the crRNA-associated nucleic acid interference complexes. Recent development in structural biology of Cas6 and Cas5d and their complexes with RNA substrates has lent new insights on principles of crRNA processing and the structural basis for linking crRNA processing to interference. Both Cas6 and Cas5d are characterized by the presence of the ferredoxin-like fold, but each has unique domain arrangement and insertion elements. Cas6 proteins often interact strongly with stable RNA stem-loop structures but can also fold unstructured RNA into stem-loop structures for their cleavage. The extraordinarily simple fold, the wide range of substrates, and kinetic properties of Cas6/Cas5d make them excellent candidates for exploring molecular evolution, protein-RNA interaction, and biotechnology applications.

| S-EPMC4286480 | biostudies-literature

Development of a genetically encodable FRET system using fluorescent RNA aptamers.

Project description:Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.

| S-EPMC5750238 | biostudies-literature

RNA aptamers that functionally interact with green fluorescent protein and its derivatives.

Project description:Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.

| S-EPMC3300005 | biostudies-literature

Effect of UV Radiation on Fluorescent RNA Aptamers' Functional and Templating Ability.

Project description:Damage from ultraviolet (UV) radiation was likely to be an important selection pressure during the origin of life. RNA is believed to have been central to the origin of life and might form the basis for simple synthetic cells. Although photodamage of DNA has been extensively studied, photodamage is highly dependent on local molecular context, and damage to functional RNAs has been relatively under-studied. We irradiated two fluorescent RNA aptamers and monitored the loss of activity, folding, and the kinetics of lesion accumulation. The loss of activity differed depending on the aptamer, with the Spinach2 aptamer retaining substantial activity after long exposure times. The binding pocket was particularly susceptible to damage, and melting of the duplex regions increased susceptibility; this is consistent with the view that duplex formation is protective. At the same time, susceptibility varied greatly depending on context, thus emphasizing the importance of studying many different RNAs to understand UV hardiness.

| S-EPMC6899979 | biostudies-literature

Ratcheting of RNA polymerase toward structural principles of RNA polymerase operations.

Project description:RNA polymerase (RNAP) performs various tasks during transcription by changing its conformational states, which are gradually becoming clarified. A recent study focusing on the conformational transition of RNAP between the ratcheted and tight forms illuminated the structural principles underlying its functional operations.

| S-EPMC4581356 | biostudies-other

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology.

Project description:RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates.

| S-EPMC6321606 | biostudies-literature

RAPID-SELEX for RNA aptamers.

Project description:Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.

| S-EPMC3869713 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data