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

Summary

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.

Abstract

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 µM theophylline in as little as 15 min.

Introduction

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 functions^2,3, amplifying said signal and improving the system'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 strategies^7,8.

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.

Protocol

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).

1. Collect 5 U of T4 polynucleotide kinase (0.5 µL, 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.
2. Add 1 µL of preprepared 5x ribozyme buffer (see Table of Materials) to each sample tube (PCR tube).
3. For the purposes of visually demonstrating specificity, add 1.5 µL 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.
4. Mix each sample tube thoroughly by pipetting 5-10 times.
5. Add 2 µL 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 µL of test solution, then mix quickly by pipetting and place on a cold block to minimize the background signal.
   ​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.
6. Once all the reaction tubes are prepared, incubate each sample (5 µL) at room temperature (23 °C) for exactly 3 min using a timer. Immediately return the sample tubes to ice (4 °C) after incubation.

2. GQ-EXPAR

1. Add 3.5 µL 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.
2. Add 31.5 µL 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.
3. Once all the samples are prepared, incubate each prepared sample (40 µL) at 55 °C for exactly 5 min using a timer. If possible, incubate on a thermocycler, although a heated lid is unnecessary.
4. Immediately return the sample tubes to ice (4 °C) after the incubation step.

3. Color development

1. Add 2 µL of Hemin solution (25 uM, see Table of Materials) to each sample tube and mix well.
2. Add 58 µL of the commercially available TMB solution (see Table of Materials) to each sample tube and mix well.
3. Incubate the color development reaction (100 µL) at room temperature for at least 3 min and up to 30 min.
   NOTE: (Optional) Color development can be stopped by adding 20 µL of 2 M sulfuric acid.
4. 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.

Representative Results

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).

Figure 1: The GQ-EXPAR system. Mechanisms of the detection and signal amplification system. Please click here to view a larger version of this figure.

Figure 2: Aptamer target and counter-target. Molecular structure of theophylline (target) and caffeine (counter-target control) as a demonstration of specificity. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

[Theophylline] (mM)	Trial 1 (A450)	Trial 2 (A450)	Trial 3 (A450)	Average (A450)
3.125	0.342	0.318	0.39	0.350 ± 0.0299
0.625	0.321	0.303	0.317	0.314 ± 0.00772
0.125	0.263	0.264	0.287	0.271 ± 0.0111
0.025	0.221	0.215	0.234	0.223 ± 0.00793
0.005	0.168	0.167	0.184	0.173 ± 0.00779
0.001	0.134	0.115	0.138	0.129 ± 0.0100
0	0.107	0.104	0.117	0.109 ± 0.00556
No Ribozyme	0.095	0.088	0.129	0.104 ± 0.0179

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. Please click here to download this File.

Supplementary Table 2: GQ-EXPAR oligonucleotide sequences. Sequences of the DNA and RNA molecules used in the detection reactions. Please click here to download this File.

Discussion

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'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.

Materials

Name	Company	Catalog Number	Comments
3,3',5,5'-Tetramethylbenzidine dihydrochloride (TMB) solution with H2O2, "MaxSignal TMB Microwell Substrate Solution"	PerkinElmer	FOOD-1806-1000	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.
5’- TCC CTC CCT CCC TCC CAG TCC AGA CTC TTC CCT CCC TCC CTC CCA GA-Biotin-3’	Integrated DNA Technologies (IDT)	Custom	Optimized QtQ47 DNA template for EXPAR to produce G-quadruplex, primed by G-quadruplex. This is used for the "exponential" part of EXPAR, to rapidly increase the amount of DNAzyme used for Step 3 color development. Template includes a 3'-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. Part of Apta-beacon Demonstration Kit Tube 2-2.
5’-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’	Integrated DNA Technologies (IDT)	Custom	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.
5’-TCC CTC CCT CCC TCC CAG TCC AGA CTC TAC GGC TTT CAC CGT TTC AAC G-Biotin-3’	Integrated DNA Technologies (IDT)	Custom	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'-biotin to prevent unintended extension by Bst 2.0 DNA polymerase during EXPAR. Part of Apta-beacon Demonstration Kit Tube 2-2.
5x Ribozyme Buffer	Aptagen, LLC	N/A	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.
Apta-beaconTM (GQ-EXPAR, TMB) Demonstration Kit	Aptagen, LLC	GQ-EXPAR-TMB	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.
Bst 2.0 DNA polymerase	New England Biolabs	M0537S	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.
Buffer 3.1	New England Biolabs	B6003SVIAL	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.
Caffeine	Sigma-Aldrich	C0750-100G	Aptamer counter-target (control), prepared with nuclease-free water. Supplied in Apta-beacon Demonstration Kit as Tube 1-1.
dNTPs	New England Biolabs	N0447S	Part of the EXPAR reaction mixture. Part of Apta-beacon Demonstration Kit Tube 2-2.
EXPAR reaction mixture	Aptagen, LLC	N/A	0.44 mM dNTPs, 0.38 μM P3tQ49, 0.38 μM 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 μg/ml BSA, 0.0635 % Tween 20. Part of Apta-beacon Demonstration Kit Tube 2-2.
Hemin	Sigma-Aldrich	H9039	Resuspended in DMF to a final concentration of 25 uM. Supplied in Apta-beacon Demonstration Kit as Tube 3-1.
Isothermal Amplification Buffer	New England Biolabs	B0537SVIAL	Combined with 2 uL of 10x Buffer 3.1 and 27.5 µL of nuclease-free water to produce 1.11x EXPAR Buffer in EXPAR Mix. Part of Apta-beacon Demonstration Kit Tube 2-2.
MgCl2	Amresco (VWR)	E525-500ML	Hammerhead ribozyme cofactor, necessary for self-cleavage. Part of Apta-beacon Demonstration Kit Tube 1-3.
MJ PTC-100 Thermocycler	MJ Research, Inc.	PTC-100	Thermocycler to control incubation temperatures. Any thermocycler or hot block can be used.
N, N-Dimethylformamide (DMF)	Sigma-Aldrich	227056-100ML	Used to resuspend hemin and maximize shelf life in freezer. Part of Apta-beacon Demonstration Kit Tube 3-1.
Nickase-polymerase Mix	Aptagen, LLC	N/A	Nt.BstNBI (9.375 units/μL) and Bst 2.0 DNA polymerase (0.5 units/μL). Supplied in Apta-beacon Demonstration Kit as Tube 2-1.
Nt.BstNBI	New England Biolabs	R0607S	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.
T4 Kinase Buffer	New England Biolabs	B0201SVIAL	Buffer for enzyme necessary to remove cyclic phosphate. Part of Apta-beacon Demonstration Kit Tube 1-4.
T4 polynucleotide kinase	New England Biolabs	M0201S	Removes cyclic phosphate post-cleavage to allow cleavage product to prime isothermal amplification reaction. Supplied in Apta-beacon Demonstration Kit as PCR Tubes.
Tecan GENios FL	Tecan	Genios-FL TWT	Plate reader to measure absorbance signal from Step 3 results.
Theophylline	Sigma-Aldrich	T1633-50G	Aptamer target, prepared with nuclease-free water. Supplied in Apta-beacon Demonstration Kit as Tube 1-2.
Tris-HCl	American Bioanalytical	AB14043-01000	Aptamer binding buffer. Part of Apta-beacon Demonstration Kit Tube 1-4.