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 (aptamer-regulated) ribozyme recognizing theophylline (see Supplementary Table 2).
<ol>
	<li>Collect 5 U of T4 polynucleotide kinase (0.5 &#181;L, see Table of Materials) in a PCR tube, which will be used to activate the ribozyme cleavage products. 
	NOTE: This component is added first so the user can see the correct dispensing of the enzyme.</li>
	<li>Add 1 &#181;L of preprepared 5x ribozyme buffer (see Table of Materials) to each sample tube (PCR tube).</li>
	<li>For the purposes of visually demonstrating specificity, add 1.5 &#181;L of 2 mM theophylline (target) or 2 mM caffeine (control) (see Table of Materials) to each sample tube as the test samples. 
	NOTE: For establishing a standard curve, it is recommended to start at 3.125 mM analyte and test five-fold dilutions until 0.001 mM.</li>
	<li>Mix each sample tube thoroughly by pipetting 5-10 times.</li>
	<li>Add 2 &#181;L of 600 nM RNA containing an aptameric domain specific to the target (see Table of Materials) to each sample tube to make a final concentration of 240 nM in 5 &#181;L of test solution, then mix quickly by pipetting and place on a cold block to minimize the background signal. 
	&#8203;NOTE: it is important to initially prepare all the reactions without ribozyme, as the self-cleavage reaction will begin immediately when ribozyme is combined with ribozyme buffer and may produce significant background.</li>
	<li>Once all the reaction tubes are prepared, incubate each sample (5 &#181;L) at room temperature (23 &#176;C) for exactly 3 min using a timer. Immediately return the sample tubes to ice (4 &#176;C) after incubation.</li>
</ol>
2. GQ-EXPAR
<ol>
	<li>Add 3.5 &#181;L of nickase-polymerase enzyme mixture (see Table of Materials) to each sample tube and mix well. 
	NOTE: The required enzyme concentrations depend on the recognition sequences, types of enzymes, and reaction temperatures, the combination of which was empirically determined.</li>
	<li>Add 31.5 &#181;L of EXPAR reaction mixture containing template, nucleotides, and reaction buffer (see Table of Materials) to each sample tube and pipet to mix. 
	NOTE: The component concentrations are optimized based on the enzymes and sequences of the polymerase products.</li>
	<li>Once all the samples are prepared, incubate each prepared sample (40 &#181;L) at 55 &#176;C for exactly 5 min using a timer. If possible, incubate on a thermocycler, although a heated lid is unnecessary.</li>
	<li>Immediately return the sample tubes to ice (4 &#176;C) after the incubation step.</li>
</ol>
3. Color development
<ol>
	<li>Add 2 &#181;L of Hemin solution (25 uM, see Table of Materials) to each sample tube and mix well.</li>
	<li>Add 58 &#181;L of the commercially available TMB solution (see Table of Materials) to each sample tube and mix well.</li>
	<li>Incubate the color development reaction (100 &#181;L) at room temperature for at least 3 min and up to 30 min. 
	NOTE: (Optional) Color development can be stopped by adding 20 &#181;L of 2 M sulfuric acid.</li>
	<li>Read the samples qualitatively by eye, or quantify the results using an absorbance plate reader at 450 nm if the color development reactions are stopped using sulfuric acid.</li>
</ol>

<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>

Aptagen, LLC manufactures and markets a GQ-EXPAR Demonstration Kit for those who want direct experience with an aptamer-based detection system.

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 enzymes required for facilitating the EXPAR and color development. Finally, considering that this is a 3-stage system, the combination of incubation times and temperatures alongside the concentrations of the reaction components meant that maximizing the signal while minimizing noise was no small challenge.
During the preparation of the ribozyme (step 1), target binding to the aptameric domain stabilizes the structure of the self-cleaving ribozyme. Despite this, the ribozyme will be capable of cleaving once introduced to the ribozyme buffer, even in the absence of a target. Thus, to minimize background signal, it is vital to keep the reactions on ice until they are ready for the defined incubation. The successful binding of the target to the aptameric domain and the resulting cleavage event produce two cleavage products, as depicted in Figure 1, stage 1: a short segment of Stem I and a newly exposed segment of Stem III with a terminal cyclic phosphate8. The T4 polynucleotide kinase present in the sample mix removes the cyclic phosphate from the Stem III cleavage product, which acts as a primer for the amplification reaction in stage 2. The concentrations and volumes of the reagents used in this system were optimized based on experiments to evaluate the background signal versus specific response4 and will need to be optimized starting from whatever point changes are made. Additionally, 3.125 mM theophylline is recommended as the highest concentration for establishing a standard curve as the color development resulting from this sample is close to the maximum absorption possible at 450 nm (Figure 4). A nonlinear regression by ordinary least squares is used to determine the standard curve for this preparation of the GQ-EXPAR system.
GQ-EXPAR, carried out in step 2, uses two DNA templates: one that is primed by the Stem III cleavage product and produces G-quadruplex, and one that is primed by G-quadruplex and produces G-quadruplex. The former reaction is more important than the latter, as translating the Stem III cleavage product into G-quadruplexes is necessary to produce a colorimetric output, while G-quadruplex self-amplification increases the signal that is already present. Bst 2.0 DNA polymerase carries out the isothermal amplification and has strand-displacement activity, while Nt. BstNBI nickase cleaves prior to the G-quadruplex sequence to allow Bst 2.0 to displace the previously generated product and produce more G-quadruplex amplicon. The buffer also supplies K+, which allows the amplification product to fold into the active G-quadruplex form. Different nickases have different recognition sequences for binding and cutting, and specific enzymes will have different cutting efficiencies; changing the sequence in the recognition/nicking site will require a change in the nickase and reoptimizing step 2.
In step 3, G-quadruplexes can function as DNAzymes with peroxidase activity, especially when associated with hemin7. The reduction of peroxide is coupled with oxidation of the substrate such as TMB to produce a detectable signal for visualization or quantification.
The key limitation in this system is the noise that the allosteric ribozyme can generate. Although the active, self-cleaving structure is not stable in the absence of the target (in this case, theophylline), the RNA has the opportunity to sample different configurations and can inadvertently access the active configuration4. This, combined with the amplification of the signal found in stage 2 and stage 3, can generate a false positive signal. It is, thus, vital to reduce the system&#39;s activity as much as possible through optimization of the RNA, buffer, and enzyme amounts, as well as minimizing the incubation times and temperatures to limit the production of RNA cleavage events that are not target-mediated.
Despite the difficulty in optimizing a GQ-EXPAR workflow, the system can be quite versatile once established. Various aptamer-controlled allosteric ribozymes have already been identified9 and can be optimized for the desired functionality5. Progress has also been made in developing allosteric DNAzymes modulated by aptamers10, expanding the chemistries that can be integrated into this platform. These options for altering the target recognition of the system have not been evaluated and would be the focus of future studies. Additionally, key components of the system can be exchanged for equivalent materials with different activity requirements, such as lower incubation temperatures for the polymerase or different recognition sequences for the nickase. The flexibility and modularity of this system allow it to adapt to a wide range of inputs.

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><tbody><tr><td>3,3&#039;,5,5&#039;-Tetramethylbenzidine dihydrochloride (TMB) solution with H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, &quot;MaxSignal TMB Microwell Substrate Solution&quot;</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.&lt;br/&gt; Supplied in Apta-beacon Demonstration Kit as Tube 3-2.</td></tr><tr><td>5&amp;rsquo;- TCC CTC CCT CCC TCC CAG TCC AGA CTC TTC CCT CCC TCC CTC CCA GA-Biotin-3&amp;rsquo;</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 &quot;exponential&quot; part of EXPAR, to rapidly increase the amount of DNAzyme used for Step 3 color development. Template includes a 3&#039;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR.&lt;br/&gt; Part of Apta-beacon Demonstration Kit Tube 2-2.</td></tr><tr><td>5&amp;rsquo;-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&amp;rsquo;</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.&lt;br/&gt; 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.&lt;br/&gt; Supplied in Apta-beacon Demonstration Kit as Tube 1-3.</td></tr><tr><td>5&amp;rsquo;-TCC CTC CCT CCC TCC CAG TCC AGA CTC TAC GGC TTT CAC CGT TTC AAC G-Biotin-3&amp;rsquo;</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&#039;-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR.&lt;br/&gt; 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 MgCl&lt;sub&gt;2&lt;/sub&gt;, 25 mM DTT. Used in Step 1.&lt;br/&gt; 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.&amp;nbsp;</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.&lt;br/&gt; 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).&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; 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 &amp;mu;M P3tQ49, 0.38 &amp;mu;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 &amp;mu;g/ml BSA, 0.0635 % Tween 20.&lt;br/&gt; 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.&lt;br/&gt; 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 &amp;micro;L of nuclease-free water to produce 1.11x EXPAR Buffer in EXPAR Mix.&lt;br/&gt; Part of Apta-beacon Demonstration Kit Tube 2-2.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Amresco (VWR)</td><td>E525-500ML</td><td>Hammerhead ribozyme cofactor, necessary for self-cleavage.&lt;br/&gt; 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.&lt;br/&gt; 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/&amp;mu;L) and Bst 2.0 DNA polymerase (0.5 units/&amp;mu;L).&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; 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.&lt;br/&gt; 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 little as 500 nM theophylline in 30 min (Figure 3)-sample preparations exposed to the target produce a blue color in step 3, while samples containing no target remain colorless. If necessary, these results can be quantified by first stopping the color development reaction as described in step 3.4, and then reading the absorbance of the resulting product at 450 nm to gauge the initial concentration of the target present. Typical results of this process are presented in Table 1, which can be fitted to a nonlinear regression to estimate quantitatively the concentration of the target present in the initial sample (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64342/64342fig01.jpg" /> 
Figure 1: The GQ-EXPAR system. Mechanisms of the detection and signal amplification system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64342/64342fig02.jpg" /> 
Figure 2: Aptamer target and counter-target. Molecular structure of theophylline (target) and caffeine (counter-target control) as a demonstration of specificity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64342/64342fig03.jpg" /> 
Figure 3: GQ-EXPAR visual results. Representative results of the system for detecting various concentrations of theophylline after 3 min and 30 min of color development. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64342/64342fig04.jpg" /> 
Figure 4: Representative theophylline standard curve. Standard curve of the system signal measured at A450 after 30 min of color development. Raw data are presented in Table 1, N = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Theophylline] (mM)</td>
			<td>Trial 1 (A450)</td>
			<td>Trial 2 (A450)</td>
			<td>Trial 3 (A450)</td>
			<td>Average (A450)</td>
		</tr>
		<tr>
			<td>3.125</td>
			<td>0.342</td>
			<td>0.318</td>
			<td>0.39</td>
			<td>0.350 &#177; 0.0299</td>
		</tr>
		<tr>
			<td>0.625</td>
			<td>0.321</td>
			<td>0.303</td>
			<td>0.317</td>
			<td>0.314 &#177; 0.00772</td>
		</tr>
		<tr>
			<td>0.125</td>
			<td>0.263</td>
			<td>0.264</td>
			<td>0.287</td>
			<td>0.271 &#177; 0.0111</td>
		</tr>
		<tr>
			<td>0.025</td>
			<td>0.221</td>
			<td>0.215</td>
			<td>0.234</td>
			<td>0.223 &#177; 0.00793</td>
		</tr>
		<tr>
			<td>0.005</td>
			<td>0.168</td>
			<td>0.167</td>
			<td>0.184</td>
			<td>0.173 &#177; 0.00779</td>
		</tr>
		<tr>
			<td>0.001</td>
			<td>0.134</td>
			<td>0.115</td>
			<td>0.138</td>
			<td>0.129 &#177; 0.0100</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0.107</td>
			<td>0.104</td>
			<td>0.117</td>
			<td>0.109 &#177; 0.00556</td>
		</tr>
		<tr>
			<td>No Ribozyme</td>
			<td>0.095</td>
			<td>0.088</td>
			<td>0.129</td>
			<td>0.104 &#177; 0.0179</td>
		</tr>
	</tbody>
</table>
Table 1: Representative theophylline standard curve raw data. Results of the system for detecting various concentrations of theophylline after 30 min of color development. No-ribozyme samples were also assessed, N = 3.
Supplementary Table 1: GQ-EXPAR setup. Summary of the GQ-EXPAR reagent volumes and order of addition as described in the protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342_Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: GQ-EXPAR oligonucleotide sequences. Sequences of the DNA and RNA molecules used in the detection reactions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64342/64342_Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>

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 ...

aptamer-based-target-detection-facilitated-3-stage-g-quadruplex

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.3K Views.  Aptagen LLC. 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. 

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

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

Genetics

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Cancer Research

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biology

Identifying G-Quadruplexes in a DNA Template Using Chemical Mapping

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Author Spotlight: Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...