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.

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ((NH4)2SO4)</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>&#160;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&#160;</td>
		</tr>
		<tr>
			<td>Heat block&#160;</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 (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</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&#160;</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 MgCl2 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 (Na2CO3)</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&#160;</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...

Watch this Scientific Journal Video about Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers at JoVE.com

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

Research

JoVE Journal

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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...