Name: determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers
Uploaded: 2022-08-09T14:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

This work was supported by the following grants to MCH: NSF-BSF 1815508 and NIH R01 GM124589. MRM was partially supported by training grant NIH T32 GM122740.

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The authors declare no conflicts of interest.

For the in vitro kinetics experiment, the same general protocol can be modified to measure the in vitro kinetics of an RNA-based fluorescent biosensor containing both a ligand-binding and fluorophore-binding domain8. In this case, the RNA should be incubated with the fluorophore prior to measurements upon injecting the ligand in order to obtain ligand response kinetics. If high variability is observed between the replicates, one can troubleshoot by checking that each sample is al...

Fluorogenic reactions are chemical reactions that generate a fluorescence signal. Fluorogenic RNA aptamers typically perform this function by binding a small molecule dye to enhance its fluorescence quantum yield (Figure 1A)1. Different fluorogenic RNA aptamer systems have been developed and consist of specific RNA aptamer sequences and the corresponding dye ligands1. Fluorogenic RNA aptamers have been appended to RNA transcripts as fluorescent tags that enable live cell imaging of mRNAs and non-coding RNAs2,3,4. They have also been placed after promoter sequences as fluorescent reporters of gene expression, similar to the use of green fluorescent protein (GFP) as a reporter, except the reporting function is at the RNA level5,6. Finally, fluorogenic RNA aptamers have been incorporated into RNA-based fluorescent biosensors, which are designed to trigger the fluorogenic reaction in response to a specific small molecule. RNA-based fluorescent biosensors have been developed for live cell imaging of various non-fluorescent metabolites and signaling molecules7,8,9,10,11.
There is growing interest in the development of fluorogenic RNA aptamers to visualize dynamic changes in RNA localization, gene expression, and small molecule signals. For each of these applications, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro kinetics for fluorogenic RNA aptamers Spinach212&#160;and Broccoli13 using a plate reader equipped with a sample injector and to determine the cellular turn-on kinetics for Spinach2 expressed in Escherichia coli using a flow cytometer. These two RNA aptamers were chosen because they have been applied to study RNA localization2,3,4, they have been used in reporters5, 6 and biosensors7,8,9,10,11, and the corresponding dye ligands (DFHBI or DFHBI-1T) are commercially available. A summary of their in vitro properties determined in the literature is given in Table 14,13,14, which informed the protocol development (e.g., the wavelengths and dye concentrations used). These results demonstrate that the fluorogenic reactions affected by RNA aptamers are rapid and should not impede accurate measurements for the desired cell biological applications.

<table><tbody><tr><td>Agarose</td><td>Thermo Fischer Scientific</td><td>BP160500</td><td></td></tr><tr><td>Agarose gel electrophoresis equipment</td><td>Thermo Fischer Scientific</td><td>B1A-BP</td><td></td></tr><tr><td>Alpha D-(+)-lactose monohydrate</td><td>Thermo Fischer Scientific</td><td>18-600-440</td><td></td></tr><tr><td>Amber 1.5 mL microcentrifuge tubes</td><td>Thermo Fischer Scientific</td><td>22431021</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>Ammonium sulfate ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>A4418</td><td></td></tr><tr><td>Attune NxT Flow cytometer</td><td>Thermo Fischer Scientific</td><td>A24861</td><td></td></tr><tr><td>Attune 1x Focusing Fluid</td><td>Thermo Fischer Scientific</td><td>A24904</td><td></td></tr><tr><td>Attune Shutdown Solution</td><td>Thermo Fischer Scientific</td><td>A24975</td><td></td></tr><tr><td>Attune Performance Tracking Beads</td><td>Thermo Fischer Scientific</td><td>4449754</td><td></td></tr><tr><td>Attune Wash Solution</td><td>Thermo Fischer Scientific</td><td>&amp;nbsp;J24974</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B6768</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>B0126</td><td></td></tr><tr><td>Carbenicillin disodium salt</td><td>Sigma-Aldrich</td><td>C3416</td><td></td></tr><tr><td>Chlorine Bleach</td><td>Amazon</td><td>B07J6FJR8D</td><td></td></tr><tr><td>Corning Costar 96-well plate</td><td>Daigger Scientific</td><td>EF86610A</td><td></td></tr><tr><td>Culture Tubes, 12 mm x 75 mm, 5 mL with attached dual position cap</td><td>Globe Scientific</td><td>05-402-31</td><td></td></tr><tr><td>DFHBI</td><td>Sigma-Aldrich</td><td>SML1627</td><td></td></tr><tr><td>DFHBI-1T</td><td>Sigma-Aldrich</td><td>SML2697</td><td></td></tr><tr><td>D-Glucose (anhydrous)</td><td>Acros Organics</td><td>AC410955000</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>DTT-RO</td><td></td></tr><tr><td>DNA loading dye</td><td>New England Biolabs</td><td>B7025S</td><td></td></tr><tr><td>DNA LoBind Tubes (2.0 mL)</td><td>Eppendorf</td><td>22431048</td><td></td></tr><tr><td>dNTPs: dATP, dCTP, dGTP, dTTP</td><td>New England Biolabs</td><td>N0446S</td><td></td></tr><tr><td>EDTA, pH 8.0</td><td>Gibco, Life Technologies</td><td>AM9260G</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Filter-tip micropipettor tips</td><td>Thermo Fischer Scientific</td><td>AM12635, AM12648, AM12655, AM12665</td><td></td></tr><tr><td>FlowJo Software</td><td>BD Biosciences</td><td>N/A</td><td>FlowJo v10 Software</td></tr><tr><td>Fluorescent plate reader with heating control</td><td>VWR</td><td>10014-924</td><td></td></tr><tr><td>Gel electrophoresis power supply</td><td>Thermo Fischer Scientific</td><td>EC3000XL2</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Glycogen AM95010</td><td>Thermo Fischer Scientific</td><td>AM95010</td><td></td></tr><tr><td>GraphPad Prism</td><td>Dotmatics</td><td>N/A</td><td>Analysis software from Academic Group License&amp;nbsp;</td></tr><tr><td>Heat block&amp;nbsp;</td><td>Thomas Scientific</td><td>1159Z11</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td></td></tr><tr><td>Inorganic pyrophosphatase</td><td>Sigma-Aldrich</td><td>I1643-500UN</td><td></td></tr><tr><td>Low Molecular Weight DNA Ladder</td><td>New England Biolabs</td><td>N3233L</td><td>Supplied with free vial of Gel Loading Dye, Purple (6x), no SDS (NEB #B7025).</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>MFCD00011110</td><td></td></tr><tr><td>Microcentrifuge tubes (1.5 mL)</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Microcentrifuge with temperature control</td><td>Marshall Scientific</td><td>EP-5415R</td><td></td></tr><tr><td>Micropipettors</td><td>Gilson</td><td>FA10001M, FA10003M, FA10005M, FA10006M</td><td></td></tr><tr><td>Micropipettor tips</td><td>Sigma-Aldrich</td><td>Z369004, AXYT200CR, AXYT1000CR</td><td></td></tr><tr><td>Millipore water filter with BioPak unit</td><td>Sigma-Aldrich</td><td>CDUFBI001, ZRQSVR3WW</td><td></td></tr><tr><td>Narrow micropipettor pipette tips</td><td>DOT Scientific</td><td>RN005R-LRS</td><td></td></tr><tr><td>PBS, 10x</td><td>Thermo Fischer Scientific</td><td>BP39920</td><td></td></tr><tr><td>PCR clean-up kit</td><td>Qiagen</td><td>28181</td><td></td></tr><tr><td>PCR primers and templates</td><td>Integrated DNA technologies</td><td></td><td></td></tr><tr><td>PCR thermocycler for thin-walled PCR tubes</td><td>Bio-Rad</td><td>1851148</td><td></td></tr><tr><td>PCR thermocycler for 0.5 mL tubes</td><td>Techne</td><td>5PRIME/C</td><td></td></tr><tr><td>pET31b-T7-Spinach2 Plasmid</td><td>Addgene</td><td>Plasmid #79783</td><td></td></tr><tr><td>Phusion High-Fidelity DNA polymerase&amp;nbsp;</td><td>New England Biolabs</td><td>M0530L</td><td>Purchase of Phusion High-Fideldity Enzyme is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, and MgCl&lt;sub&gt;2&lt;/sub&gt; and DMSO solutions.</td></tr><tr><td>Polyacrylamide gel electrophoresis gel comb, C.B.S. Scientific</td><td>C.B.S. Scientific</td><td>VGC-1508</td><td></td></tr><tr><td>Polyacrylamide gel electrophoresis equipment</td><td>C.B.S. Scientific</td><td>ASG-250</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Razor blades</td><td>Genesee Scientific</td><td>38-101</td><td></td></tr><tr><td>rNTPs: ATP, CTP, GTP, UTP</td><td>New England Biolabs</td><td>N0450L</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Short wave UV light source</td><td>Thermo Fischer Scientific</td><td>11758221</td><td></td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S7795</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Sodium phosphate dibasic, anhydrous</td><td>Thermo Fischer Scientific</td><td>S375-500</td><td></td></tr><tr><td>SoftMax Pro</td><td>Molecular Devices</td><td>N/A</td><td>SoftMax Pro 6.5.1 (platereader software) obtained through Academic Group License</td></tr><tr><td>Sterile filter units</td><td>Thermo Fischer Scientific</td><td>09-741-88</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Thermo Fischer Scientific</td><td>S33102</td><td></td></tr><tr><td>TAE buffer for agarose gel electrophoresis</td><td>Thermo Fischer Scientific</td><td>AM9869</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>TRIS-RO</td><td></td></tr><tr><td>Tryptone (granulated)</td><td>Thermo Fischer Scientific</td><td>M0251S</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>New England Biolabs</td><td>M0251S</td><td></td></tr><tr><td>Urea-PAGE Gel system&amp;nbsp;</td><td>National Diagnostics</td><td>EC-833</td><td></td></tr><tr><td>UV fluorescent TLC plate</td><td>Sigma-Aldrich</td><td>1.05789.0001</td><td></td></tr><tr><td>UV/Vis spectrophotometer</td><td>Thermo Fischer Scientific</td><td>ND-8000-GL</td><td></td></tr><tr><td>Vortex mixer</td><td>Thermo Fischer Scientific</td><td>2215415</td><td></td></tr><tr><td>Xylene cyanol</td><td>Sigma-Aldrich</td><td>X4126</td><td></td></tr><tr><td>Yeast Extract (Granulated)</td><td>Thermo Fischer Scientific</td><td>BP9727-2</td><td></td></tr></tbody></table>

determination of in vitro and cellular turn-on kinetics for fluorogenic rna aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s&#8722;1 and 0.35 s&#8722;1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

In vitro kinetics 
The sequences of the DNA templates and primers, which are purchased as synthetic oligonucleotides, are shown in Table 2, and the reagent recipes are shown in Supplementary File 1. PCR amplification is used to scale up the amount of DNA template with the T7 promoter, which is required for the subsequent in vitro transcription (IVT) reaction. In addition, PCR amplification can be used for two purposes in the same r...

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Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

determination-vitro-cellular-turn-on-kinetics-for-fluorogenic-rna

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Biochemistry

1.7K Views.  University of Utah. The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

Video: Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetics

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Biology

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

Bioengineering

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Summary

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Chapters in this video

In Vitro Transcription Assays and Their Application in Drug Discovery

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Measuring the Kinetics of mRNA Transcription in Single Living Cells

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo