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.
