Name: aptamer-based target detection facilitated by a 3-stage g-quadruplex isothermal exponential amplification reaction
Uploaded: 2022-10-06T13:40:00
Description: Watch this Scientific Journal Video about Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction at JoVE.com

This research was supported by the Research and Development Funding from Aptagen LLC.

See Supplementary Table 1 (the reaction set-up table) for tube preparation, including the specific volumes and concentrations of the reaction components. The protocol demonstrated here uses a preassembled detection platform kit as described in the Table of Materials. All the components must be kept on ice unless indicated otherwise.
1. Preparation of ribozyme
NOTE: The primary detection component is an allosteric (apt...

<ol>
	<li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346 (6287), 818-822 (1990).</li><li>Tang, J., Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+allosteric+ribozymes.">Rational design of allosteric ribozymes.</a> Chemical Biology. 4 (6), 453-459 (1997).</li><li>Stoltenburg, R., Reinemann, C., Strehlitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SELEX--A+(r)evolutionary+method+to+generate+high-affinity+nucleic+acid+ligands.">SELEX--A (r)evolutionary method to generate high-affinity nucleic acid ligands.</a> Biomolecular Engineering. 24 (4), 381-403 (2007).</li><li>Liao, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+colorimetric+system+for+detecting+target+antigens+by+a+three-stage+signal+transformation-amplification+strategy.">A simple colorimetric system for detecting target antigens by a three-stage signal transformation-amplification strategy.</a> Biochemistry. 57 (34), 5117-5126 (2018).</li><li>Soukup, G. A., Emilsson, G. A., Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+molecular+recognition+of+RNA+aptamers+by+allosteric+selection.">Altering molecular recognition of RNA aptamers by allosteric selection.</a> Journal of Molecular Biology. 298 (4), 623-632 (2000).</li><li>Van Ness, J., Van Ness, L. K., Galas, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+reactions+for+the+amplification+of+oligonucleotides.">Isothermal reactions for the amplification of oligonucleotides.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (8), 4504-4509 (2003).</li><li>Cheng, X., Liu, X., Bing, T., Cao, Z., Shangguan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+peroxidase+activity+of+G-quadruplex-hemin+complexes+and+its+application+in+ligand+screening.">General peroxidase activity of G-quadruplex-hemin complexes and its application in ligand screening.</a> Biochemistry. 48 (33), 7817-7823 (2009).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter-triggered+isothermal+exponential+amplification+strategy+in+ultrasensitive+homogeneous+label-free+electrochemical+nucleic+acid+biosensing.">Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing.</a> Chemical Communications. 50 (47), 6211-6213 (2014).</li><li>Tabuchi, T., Yokobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+riboswitches.">Cell-free riboswitches.</a> RSC Chemical Biology. 2 (5), 1430-1440 (2021).</li><li>Ao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+an+expression+platform+in+the+SELEX+cycle+to+select+DNA+aptamer+binding+to+a+disease+biomarker.">Integration of an expression platform in the SELEX cycle to select DNA aptamer binding to a disease biomarker.</a> ACS Omega. 7 (12), 10804-10811 (2022).</li></ol>

The method presented here takes advantage of the transition between an initially disordered secondary structure in an allosteric ribozyme and the additional stability conferred through the binding of the target to the aptameric domain to activate the cis-cleaving hammerhead ribozyme. The stability of the ribozyme was adjusted to minimize catalytic activity in the absence of the target while allowing target binding to restore the active structure. Additionally, care must be taken to balance the buffers of the multiple enz...

Aptamers are typically single-stranded DNA or RNA selected through an evolutionary process with high binding affinity and specificity to the desired targets1. In addition to binding ability, aptamers can be linked to and control motifs with signal-output functions2,3, amplifying said signal and improving the system&#39;s sensitivity. The G-quadruplex isothermal exponential amplification reaction (GQ-EXPAR) system is a three-part system (Figure 1) that develops a visual signal as successive components are added to a single reaction vessel to produce a visual output4. This system allows for the detection of a specific target, herein theophylline, in a given sample within 15 min using a streamlined workflow to allow for fast, specific detection of a target of interest. This method must be considered for samples where specific quantification of the target concentration is a lower concern than high specificity of the response in a short amount of time.
An allosteric riboswitch, a structure-switching RNA molecule (ribozyme) that undergoes self-cleavage, produces the initial signal. This construct is based on the hammerhead ribozyme, with an aptameric domain introduced in Stem II as a regulator of cleavage activity. Its self-cleavage function is activated when its aptameric domain is stabilized on binding to its target5. Otherwise, the switch is inactive in its native state.
The subsequent exponential amplification reaction (EXPAR) uses the release of the self-cleaved RNA strand from the first stage to prime an isothermal amplification reaction6. The amplification product of EXPAR has peroxidase activity7, acting as the basis for the last stage of the system. When some substrates are oxidized in conjunction with peroxide breakdown, they produce a fluorescent output that can be measured on various instrumentation. Other common substrates can be substituted to produce a colored product for visual detection. EXPAR and the peroxidase activity of its amplification products act as a 2-stage signal-enhancer, increasing sensitivity to greater levels compared to conventional strategies7,8.
The detection of theophylline versus caffeine is used as an example of the specificity of this detection platform, as they differ by only a single methyl group (Figure 2). This demonstration of the system produces a colorimetric output for visual detection of at least 500 nM theophylline.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,3&#39;,5,5&#39;-Tetramethylbenzidine dihydrochloride (TMB) solution with H2O2, &#34;MaxSignal TMB Microwell Substrate Solution&#34;</td>
			<td>PerkinElmer</td>
			<td>FOOD-1806-1000</td>
			<td>Color development reagent. Minimize exposure to light and atmosphere. Previously BIOO Scientific catalog number 1806. 
			Supplied in Apta-beacon Demonstration Kit as Tube 3-2.</td>
		</tr>
		<tr>
			<td>5&#8217;- TCC CTC CCT CCC TCC CAG TCC AGA CTC TTC CCT CCC TCC CTC CCA GA-Biotin-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Optimized QtQ47 DNA template for EXPAR to produce G-quadruplex, primed by G-quadruplex. This is used for the &#34;exponential&#34; part of EXPAR, to rapidly increase the amount of DNAzyme used for Step 3 color development. Template includes a 3&#39;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>5&#8217;-GGG AAC UAU ACA ACC UAG GGC GAC CCU GAU GAG CCU UAU ACC AGC CGA AAG GCC CUU GGC AGA CGU UGA AAC GGU GAA AGC CGU AGG UUG CCC UAG GUU GUA UAG UU-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Theophylline-recognizing allosteric ribozyme sequence (self-cleaving ribozyme regulated by RNA aptamer domain for theophylline) modified from Soukup et al. 
			Soukup, G. A., Emilsson, G. A., and Breaker, R. R. (2000) Altering molecular recognition of RNA aptamers by allosteric selection, Journal of molecular biology 298, 623-632. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-3.</td>
		</tr>
		<tr>
			<td>5&#8217;-TCC CTC CCT CCC TCC CAG TCC AGA CTC TAC GGC TTT CAC CGT TTC AAC G-Biotin-3&#8217;</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>Custom</td>
			<td>Optimized P3tQ49 DNA template for EXPAR to produce G-quadruplex, primed by P3 cleavage product. This is used in Step 2 to translate the cleavage product into DNAzyme used for Step 3 color development. Template includes a 3&#39;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>5x Ribozyme Buffer</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>5x composition: 600 mM Tris-HCl (pH 7.5), 150 mM MgCl2, 25 mM DTT. Used in Step 1. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-4.</td>
		</tr>
		<tr>
			<td>Apta-beaconTM (GQ-EXPAR, TMB) Demonstration Kit</td>
			<td>Aptagen, LLC</td>
			<td>GQ-EXPAR-TMB</td>
			<td>The demo kit showcases the specificity of the colorimetric assay by detecting difficult small molecule targets, theophylline versus caffeine, which only differ by a single methyl group.&#160;</td>
		</tr>
		<tr>
			<td>Bst 2.0 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0537S</td>
			<td>Isothermal amplification polymerase with strand-displacement activity. Part of the Nickase-Polymerase Mix, prepared at 9.375 U/uL. 
			Part of Apta-beacon Demonstration Kit Tube 2-1.</td>
		</tr>
		<tr>
			<td>Buffer 3.1</td>
			<td>New England Biolabs</td>
			<td>B6003SVIAL</td>
			<td>Combined with 2 uL of 10X Isothermal Amplification Buffer and 27.5 uL of nuclease-free water to produce 1.11X EXPAR Buffer in EXPAR Mix. This product replaces the previously-used B7203SVIAL that was part of the initial system development (same composition except non-recombinant BSA). 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma-Aldrich</td>
			<td>C0750-100G</td>
			<td>Aptamer counter-target (control), prepared with nuclease-free water. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-1.</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0447S</td>
			<td>Part of the EXPAR reaction mixture. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>EXPAR reaction mixture</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>0.44 mM dNTPs, 0.38 &#956;M P3tQ49, 0.38 &#956;M QtQ47, 44.44 mM Tris-HCl (pH 8.4), 63.5 mM NaCl, 31.5 mM KCl, 6.35 mM MgCl2, 1.27 mM MgSO4, 6.35 mM (NH4)2SO4, 63.5 &#956;g/ml BSA, 0.0635 % Tween 20. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma-Aldrich</td>
			<td>H9039</td>
			<td>Resuspended in DMF to a final concentration of 25 uM. 
			Supplied in Apta-beacon Demonstration Kit as Tube 3-1.</td>
		</tr>
		<tr>
			<td>Isothermal Amplification Buffer</td>
			<td>New England Biolabs</td>
			<td>B0537SVIAL</td>
			<td>Combined with 2 uL of 10x Buffer 3.1 and 27.5 &#181;L of nuclease-free water to produce 1.11x EXPAR Buffer in EXPAR Mix. 
			Part of Apta-beacon Demonstration Kit Tube 2-2.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Amresco (VWR)</td>
			<td>E525-500ML</td>
			<td>Hammerhead ribozyme cofactor, necessary for self-cleavage. 
			Part of Apta-beacon Demonstration Kit Tube 1-3.</td>
		</tr>
		<tr>
			<td>MJ PTC-100 Thermocycler</td>
			<td>MJ Research, Inc.</td>
			<td>PTC-100</td>
			<td>Thermocycler to control incubation temperatures. Any thermocycler or hot block can be used.</td>
		</tr>
		<tr>
			<td>N, N-Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056-100ML</td>
			<td>Used to resuspend hemin and maximize shelf life in freezer. 
			Part of Apta-beacon Demonstration Kit Tube 3-1.</td>
		</tr>
		<tr>
			<td>Nickase-polymerase Mix</td>
			<td>Aptagen, LLC</td>
			<td>N/A</td>
			<td>Nt.BstNBI (9.375 units/&#956;L) and Bst 2.0 DNA polymerase (0.5 units/&#956;L). 
			Supplied in Apta-beacon Demonstration Kit as Tube 2-1.</td>
		</tr>
		<tr>
			<td>Nt.BstNBI</td>
			<td>New England Biolabs</td>
			<td>R0607S</td>
			<td>Nicking enzyme to allow continued isothermal amplification. Part of the Nickase-Polymerase Mix, prepared at 0.5 U/uL. 
			Part of Apta-beacon Demonstration Kit Tube 2-1.</td>
		</tr>
		<tr>
			<td>T4 Kinase Buffer</td>
			<td>New England Biolabs</td>
			<td>B0201SVIAL</td>
			<td>Buffer for enzyme necessary to remove cyclic phosphate. 
			Part of Apta-beacon Demonstration Kit Tube 1-4.</td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td>Removes cyclic phosphate post-cleavage to allow cleavage product to prime isothermal amplification reaction. 
			Supplied in Apta-beacon Demonstration Kit as PCR Tubes.</td>
		</tr>
		<tr>
			<td>Tecan GENios FL</td>
			<td>Tecan</td>
			<td>Genios-FL TWT</td>
			<td>Plate reader to measure absorbance signal from Step 3 results.</td>
		</tr>
		<tr>
			<td>Theophylline</td>
			<td>Sigma-Aldrich</td>
			<td>T1633-50G</td>
			<td>Aptamer target, prepared with nuclease-free water. 
			Supplied in Apta-beacon Demonstration Kit as Tube 1-2.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>American Bioanalytical</td>
			<td>AB14043-01000</td>
			<td>Aptamer binding buffer. 
			Part of Apta-beacon Demonstration Kit Tube 1-4.</td>
		</tr>
	</tbody>
</table>

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 detection platform depicted in Figure 1 converts aptameric target recognition into visually distinct differences between the sample preparations (target vs. non-target, Figure 2) in a short period of time. An allosteric ribozyme identified by Soukup et al.5 served as the starting point for creating a less noisy sequence with response to the target over the control and negative samples. The optimized construct was able to recognize as ...

Watch this Scientific Journal Video about Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction at JoVE.com

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

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 ...

This method offers a way to amplify the signal from binding events into a form that is detectable visually for low resource use or can be quantified using absorbance-based instrumentation. This is a relatively quick, one-pot protocol to determine the presence of the aptamer's target in a sample of interest. Results can be compared against a standard calibration curve if quantification is desired.Individuals must be very precise when transferring and mixing the solutions, as insufficient mixing will lead to negative results. Also, ensure reagents and solutions are kept chilled until use to minimize background signal. Begin by activating the ribozyme cleavage products by adding five units of T4 polynucleotide kinase in a PCR tube.Then, add one microliter of pre-prepared 5X ribozyme buffer to each sample tube. To visually demonstrate specificity, add 1.5 microliters of two millimolar theophylline or two millimolar caffeine to each sample tube as the test samples and mix each sample thoroughly by pipetting five to 10 times. Next, add two microliters of 600 nanomolar RNA containing a target-specific aptameric domain to each sample tube, followed by pipetting to mix the components quickly.Now, place the tube on a cold block to minimize the background signal. After preparing all the reaction tubes, incubate each sample at room temperature for precisely three minutes. At the end of the incubation, return the sample tubes to ice immediately.Add 3.5 microliters of nicase polymerase enzyme mixture to each sample tube and mix well. Then, add 31.5 microliters of EXPAR reaction mixture containing template nucleotides and reaction buffer to each sample tube and mix by pipetting. Once all the samples are prepared, incubate the prepared sample at 55 degrees Celsius for five minutes using a timer.Immediately return the sample tubes to the ice after incubation. Add two microliters of hemin solution to each sample tube for color development and mix well. Then, add 58 microliters of the commercially available TMB solution to each sample tube, mix well, and incubate for color development.Read the samples qualitatively by eye or quantify the results using an absorbance plate reader, as described in the manuscript. The optimized construct could recognize as little as 500 nanomolar theophylline in 30 minutes. Sample preparations exposed to the target produce a blue color, while samples containing no target remain colorless.A standard curve was prepared from the signal measured at A450 after 30 minutes of color development. It is important to keep samples on ice when not carrying out incubations, as even the optimized RNA will still exhibit a low background activity level.

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