The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold
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Truong, Lynda↗
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Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET
To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor–acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner. Fluorescence turn-on RNA aptamers are genetically encodable tags that appear to rigidly confine their cognate fluorophores, and thus have the potential to report angular-resolved FRET. In this work, we use the fluorescent aptamers Broccoli and Mango-III as donor and acceptor, respectively, to measure the angular dependence of FRET. Joining the two fluorescent aptamers by a helix of variable length allowed systematic rotation of the acceptor fluorophore relative to the donor. FRET oscillated in a sinusoidal manner as a function of helix length, consistent with simulated data generated from models of oriented fluorophores separated by an inflexible helix. Analysis of the orientation dependence of FRET allowed us to demonstrate structural rigidification of the NiCo riboswitch upon transition metal-ion binding. This application of fluorescence turn-on aptamers opens the way to improved structural interpretation of ensemble and single-molecule FRET measurements of RNA.
59 BASIC BIOLOGICAL SCIENCES↗
Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair
Abstract not provided
Trachman, III, Robert J.↗
Handheld Fluorescence Resonance Energy Transfer (FRET)-Aptamer Sensor for Bone Markers
Astronauts lose significant bone mass during lengthy space flights. NASA wishes to monitor this bone loss in order to develop nutritional and exercise countermeasures. Operational Technologies Corporation (OpTech) has developed a handheld device that quantifies bone loss in a spacecraft environment. The innovation works by adding fluorescent dyes and quenchers to aptamers to enable pushbutton, one-step bind-and-detect FRET assays that can be freeze-dried, rehydrated with body fluids, and used to quantify bone loss.
Bruno, John G.↗
Structural basis for ring-opening fluorescence by the RhoBAST RNA aptamer
Abstract Tagging RNAs with fluorogenic aptamers has enabled imaging of transcripts in living cells, revealing novel aspects of RNA metabolism and dynamics. While a diverse set of fluorogenic aptamers has been developed, a new generation of aptamers uses the ring-opening of spirocyclic rhodamine dyes to achieve robust performance in live mammalian cells. These fluorophores have two chemical states: a cell-permeable spirocyclic state and a fluorescent zwitterionic state. The SpyRho555 dye is mostly non-fluorescent in solution and becomes fluorescent in complex with the RhoBAST aptamer. To understand the basis for RhoBAST-SpyRho555 fluorogenicity, we have determined crystal structures of RhoBAST in complex with 5-carboxytetramethylrhodamine and a SpyRho555 analogue, MaP555. RhoBAST is organized by a perfect four-way junction that positions two loops to form the dye-binding pocket. The core of the ligand resides between a tri-adenine floor and a single guanine base, largely driven by π-stacking interactions. Importantly, the unpaired guanine interacts with the 3-position group of MaP555 to stabilize the open conformation, supported by mutagenesis data, and may play an active role in promoting the open conformation of the dye. This work has implications for the development of new fluorogenic aptamers with improved properties using structure-guided design approaches.
Biochemistry & Molecular Biology↗
Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST
The fluorescent light-up aptamer RhoBAST, which binds and activates the fluorophore–quencher conjugate tetramethylrhodamine-dinitroaniline with high affinity, super high brightness, remarkable photostability, and fast exchange kinetics, exhibits excellent performance in super-resolution RNA imaging. Here we determine the co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline to elucidate the molecular basis for ligand binding and fluorescence activation. The structure exhibits an asymmetric “A”-like architecture for RhoBAST with a semi-open binding pocket harboring the xanthene of tetramethylrhodamine at the tip, while the dinitroaniline quencher stacks over the phenyl of tetramethylrhodamine instead of being fully released. Molecular dynamics simulations show highly heterogeneous conformational ensembles with the contact-but-unstacked fluorophore–quencher conformation for both free and bound tetramethylrhodamine-dinitroaniline being predominant. The simulations also show that, upon RNA binding, the fraction of xanthene-dinitroaniline stacked conformation significantly decreases in free tetramethylrhodamine-dinitroaniline. This highlights the importance of releasing dinitroaniline from xanthene tetramethylrhodamine to unquench the RhoBAST–tetramethylrhodamine-dinitroaniline complex. Using SAXS and ITC, we characterized the magnesium dependency of the folding and binding mode of RhoBAST in solution and indicated its strong structural robustness. The structures and binding modes of relevant fluorescent light-up aptamers are compared, providing mechanistic insights for rational design and optimization of this important fluorescent light-up aptamer-ligand system.
59 BASIC BIOLOGICAL SCIENCES↗
FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D
Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is called a "lights on" FRET response. The vitamin D aptamer beacon gives a "lights off" or inversely proportional fluorescence response to the amount of vitamin D present in diluted serum. These FRET-aptamer assays are rapid (<30 minutes), sensitive (low ng/mL detection limits), and quite easy to carry out (add sample, mix, and detect in the handheld reader). Benefits include the speed of the assays as well as the small amount of space taken up by the handheld reader and cuvette assays. The aptamer DNA sequences represent novel additional features of the existing (patent-pending) FRET-aptamer assay platform.
Bruno, John G.↗
Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers
Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. In this study, we aim to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.
60 APPLIED LIFE SCIENCES↗
Metastable multimeric G-quadruplex 2′FY-RNA aptamers that selectively bind pyoverdines
Two 2′FY-RNA aptamers with distinct sequences were selected for specific binding to pyoverdine-Pf5 (PVD-Pf5), increasing chromophore fluorescence upon binding. They also recognized the peptide portion of pyoverdines, as shown by their differential specificity for related variants. Computational analysis and experimental data (NMM binding, CD spectra) identified G-quadruplex structures that were thermally metastable but reformed in the presence of PVD-Pf5. Further structural studies mainly with one aptamer revealed imino proton peaks in 1D H-NMR and pressure stability up to 2 kbar. Electrophoretic evidence identified dimeric G-quadruplexes formed by the 2′FY-RNA aptamers and their RNA equivalents. While cations were necessary for PVD-Pf5 binding, they were not required for G-quadruplex formation. Given the established role of G-quadruplexes as protein interaction sites, multimeric G-quadruplexes offer a potential framework for structure-based regulatory mechanisms in cellular RNAs. In addition to previously characterized multimeric G-quadruplexes, these aptamers contribute novel sequences that expand the repertoire of known multimeric G-quadruplexes.
2′FY-RNA↗
Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads
Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Nanoscale Test Strips for Multiplexed Blood Analysis
A critical component of the DNA Medicine Institute's Reusable Handheld Electrolyte and Lab Technology for Humans (rHEALTH) sensor are nanoscale test strips, or nanostrips, that enable multiplexed blood analysis. Nanostrips are conceptually similar to the standard urinalysis test strip, but the strips are shrunk down a billionfold to the microscale. Each nanostrip can have several sensor pads that fluoresce in response to different targets in a sample. The strips carry identification tags that permit differentiation of a specific panel from hundreds of other nanostrip panels during a single measurement session. In Phase I of the project, the company fabricated, tested, and demonstrated functional parathyroid hormone and vitamin D nanostrips for bone metabolism, and thrombin aptamer and immunoglobulin G antibody nanostrips. In Phase II, numerous nanostrips were developed to address key space flight-based medical needs: assessment of bone metabolism, immune response, cardiac status, liver metabolism, and lipid profiles. This unique approach holds genuine promise for space-based portable biodiagnostics and for point-of-care (POC) health monitoring and diagnostics here on Earth.
Chan, Eugene↗
Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture
Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.
59 BASIC BIOLOGICAL SCIENCES↗
NASA Tech Briefs, September 2013
Topics include: ISS Ammonia Leak Detection Through X-Ray Fluorescence; A System for Measuring the Sway of the Vehicle Assembly Building; Fast, High-Precision Readout Circuit for Detector Arrays; Victim Simulator for Victim Detection Radar; Hydrometeor Size Distribution Measurements by Imaging the Attenuation of a Laser Spot; Quasi-Linear Circuit; High-Speed, High-Resolution Time-to-Digital Conversion; Li-Ion Battery and Supercapacitor Hybrid Design for Long Extravehicular Activities; Ultrasonic Low-Friction Containment Plate for Thermal and Ultrasonic Stir Weld Processes; High-Powered, Ultrasonically Assisted Thermal Stir Welding; Next-Generation MKIII Lightweight HUT/Hatch Assembly; Centrifugal Sieve for Gravity-Level-Independent Size; Segregation of Granular Materials; Ion Exchange Technology Development in Support of the Urine Processor Assembly; Nickel-Graphite Composite Compliant Interface and/or Hot Shoe Material; UltraSail CubeSat Solar Sail Flight Experiment; Mechanism for Deploying a Long, Thin-Film Antenna From a Rover; Counterflow Regolith Heat Exchanger; Acquisition and Retaining Granular Samples via a Rotating Coring Bit; Very-Low-Cost, Rugged Vacuum System; Medicine Delivery Device With Integrated Sterilization and Detection; FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D; Multimode Directional Coupler for Utilization of Harmonic Frequencies from TWTAs; Dual-Polarization, Multi-Frequency Antenna Array for use with Hurricane Imaging Radiometer; Complementary Barrier Infrared Detector (CBIRD) Contact Methods; Autonomous Control of Space Nuclear Reactors; High-Power, High-Speed Electro-Optic Pockels Cell Modulator; Covariance Analysis Tool (G-CAT) for Computing Ascent, Descent, and Landing Errors; Enigma Version 12; Micrometeoroid and Orbital Debris (MMOD) Shield Ballistic Limit Analysis Program; Spitzer Telemetry Processing System; Planetary Protection Bioburden Analysis Program; Wing Leading Edge RCC Rapid Response Damage Prediction Tool (IMPACT2); ISSM: Ice Sheet System Model; Automated Loads Analysis System (ATLAS); Integrated Main Propulsion System Performance Reconstruction Process/Models. Phoenix Telemetry Processor; Contact Graph Routing Enhancements Developed in ION for DTN; GFEChutes Lo-Fi; Advanced Strategic and Tactical Relay Request Management for the Mars Relay Operations Service; Software for Generating Troposphere Corrections for InSAR Using GPS and Weather Model Data; Ionospheric Specifications for SAR Interferometry (ISSI); Implementation of a Wavefront-Sensing Algorithm; Sally Ride EarthKAM - Automated Image Geo-Referencing Using Google Earth Web Plug-In; Trade Space Specification Tool (TSST) for Rapid Mission Architecture (Version 1.2); Acoustic Emission Analysis Applet (AEAA) Software; Memory-Efficient Onboard Rock Segmentation; Advanced Multimission Operations System (ATMO); Robot Sequencing and Visualization Program (RSVP); Automating Hyperspectral Data for Rapid Response in Volcanic Emergencies; Raster-Based Approach to Solar Pressure Modeling; Space Images for NASA JPL Android Version; Kinect Engineering with Learning (KEWL); Spacecraft 3D Augmented Reality Mobile App; MPST Software: grl_pef_check; Real-Time Multimission Event Notification System for Mars Relay; SIM_EXPLORE: Software for Directed Exploration of Complex Systems; Mobile Timekeeping Application Built on Reverse-Engineered JPL Infrastructure; Advanced Query and Data Mining Capabilities for MaROS; Jettison Engineering Trajectory Tool; MPST Software: grl_suppdoc; PredGuid+A: Orion Entry Guidance Modified for Aerocapture; Planning Coverage Campaigns for Mission Design and Analysis: CLASP for DESDynl; and Space Place Prime.
Source record↗
RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring
Abstract Background Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. Results We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO 2 and NaHCO 3 , and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO 2 were 5–7 times higher than the ones grown with 20 mM NaHCO 3 , at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO 3 − to CO 2 . The highest RuBisCO activity of 2.13 nmol of NAD + / µg of Chl-a/ min was observed with 50 µg of protein and 5% CO 2 . Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited ~ 3.4-fold slower reaction rate (0.37 min −1 ) than the biochemical assay when using 5% CO 2 . We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO 3 as compared to the cells that were grown without NaHCO 3 supplementation. Conclusions Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO 2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO 3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO 2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO 2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.
59 BASIC BIOLOGICAL SCIENCES↗