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

The overall goal of the following experiment is to generate structure switching apti for integration as recognition elements on detection platforms that function by signaling a change in aptamer confirmation upon target binding. This is achieved by adding a biotinylated capture probe and complimentary DNA library to strep tabin coated magnetic beads to immobilize the DNA library on the bead surface. Next, a magnet is applied to the solution to partition the binding sequences from the non-binding sequences remaining on the beads.

The retained sequences are then purified and amplified for the next round of selection. After a few rounds of selection, a negative selection step is added into the process. After several rounds of selection, the selected aptr demonstrates a structural change upon target binding based on a gold nanoparticle target detection assay.

The made advantage of this technique over existing techniques like solution-based cell X, is that we don't have to immobilize the target to a solid support. So the implications of this technique are in the risk assessment of the physiological state of an individual, and it's due to the fact that cortisol is an indicator of stress, which can lead to the assessment of the of health issues. Always use freshly prepared DNA solutions for this protocol, specifically for the target and control.

The preparation of the DNA requires special attention. Transfer a 100 microliter aliquot of the DNA library into a thermocycler tube and heat it at 95 degrees Celsius for five minutes in a thermo mixer. Then transfer the tube to ice until it is needed before using this DNA determine its exact concentration on a spectrophotometer.

Next, prepare the magnetic beads, vortex strept encoded magnetic beads, and transfer 400 microliters of them to a new micro centrifuge tube. Place this tube on the magnet for two minutes and remove the supernatant, then wash the beads. Add 400 microliters of binding buffer and vortex them to complete the wash.

Use a magnet to separate the beads and discard the supernatant. Repeat this wash process three times. Next, immobilize the capture probe on the magnetic beads.

Start by Resus suspending the beads and 400 microliters of binding buffer with 50 micromolar capture probe. Then incubate the beads for 10 minutes with gentle agitation at room temperature. To remove the free capture probe, wash the beads three more times as before, using 400 microliters of binding buffer per wash.

After the three washes, add another 400 microliters of buffer and collect 100 microliters of bead solution for the remaining steps while saving the remainder at four degrees Celsius. The remainder can be used for subsequent rounds of selection for up to three weeks. When more beads are needed, they can be prepared with longer probes for increased stringency in this selection scheme.

For example, longer probes were used after round 13, at which point significant enrichment has been achieved using other stringencies. When using beads taken from storage, begin by washing the working aliquot twice, using 200 microliters of binding buffer per wash. Otherwise, freshly made beads are already clean.

Now add 100 microliters of snap cool DNA to the beads and incubate them for half an hour at room temperature with gentle agitation. After the incubation, isolate the beads before discarding the supernatant. Calculate the concentration of DNA contains to determine how much DNA bound to the beads.

Next, wash the beads three times with 200 microliters of binding buffer. Let the binding buffer wash the beads with gentle agitation for five minutes each time. Then prepare the target molecule in binding buffer at 100 micromolar and resuspend the beads in 200 microliters of this preparation, the target molecule.

In this case, cortisol is typically highly concentrated in the first round of selection. Incubate the beads with the target for half an hour with gentle agitation at ambient conditions. After the incubation, save the snat containing the target alluded sequences.

After every other round of enrichment, take a sample from the final supernatant to monitor the process for monitoring. Remove the cortisol from the supernatant by dialysis. This can be skipped if the target molecule does not interfere with measuring DNA absorbance at 260 nanometers after every round, amplify the target binding DNA by PCR using the collected supernatant from the positive selection step as a template.

Preliminarily always run a small test PCR to determine the optimal number of amplifications. Take 10 microliter samples after each cycle and compare the samples to a 25 base pair ladder. Here, the library should run at 85 base pairs over amplified products are seen after the 10th cycle, so fewer cycles should be used.

This result can vary from round to round, so a test run is always necessary. Then perform a large scale PCR using the selected number of cycles using eight strip tubes. Amplify six to eight milliliters of reaction mixture to get the required amount of amplicon for the next round of selection, which is between one and 2.5 nanomoles of DNA.

Double check a sample of the PCR products using a gel. Then convert the bulk of the products to single stranded DNA using non-magnetic strept coated beads. Load 300 microliters of beads into a small column blocked by fritz.

Then using a lure lock syringe, gently wash the beads with five milliliters of binding buffer. When changing the solution to wash the column, remove the syringe from the column and remove the plunger from the syringe. This prevents unnecessary disturbance of the beads.

Create and wash enough bead columns for one milliliter of PCR product per column. Pass a PCR product through the columns using gentle pressure. The DNA products will be retained by the beads attached by their antisense strands.

Then wash the DNA bound columns with five milliliters of binding buffer to remove all the PCR components, leaving only the binding pool DNA to de hybridize the DNA from the column add 0.5 milliliters of 0.2 molar sodium hydroxide. Then desalt the single stranded DNA in the eit using a desalting column prepared with nuclease free water. Discard the initial 0.5 milliliters of flow through.

Then pass one milliliter of nuclease free water through the column to collect the desalted DNA. Measure the absorbance of the collected DNA and prepare it for the next round of selection. As described in the text protocol, the collected single-stranded DNA is now added back to a freshly prepared aliquot of beads prepared with capture probe.

The second round of selection is similar to the first with a lower cortisol concentration employed. However, beginning in the third round, incorporate a negative selection step before the positive selection. For the first round of negative selection, add 200 microliters of one micromolar progesterone in binding buffer to the beads.

It is structurally similar but distinct from cortisol. In subsequent rounds, increase the concentration of progesterone after gently shaking the mixture for half an hour at room temperature. Discard the supinate and wash the beads two times using 200 microliters of the binding buffer.

Then proceed with positive selection. As previously described as the rounds progress, the concentration of the substrate varied to affect the stringency of the selection process. Details are discussed in the text protocol.

Next, follow the text protocol to perform the gold nanoparticle assay for cortisol detection. This rapid and straightforward color metric readout relies on a structural change of the aptamer upon target binding. Thus, it provides a metric to assess the ability of the method to select abers with structure switching properties.

Another advantage to the gold nanoparticle assay is that it amplifies a signal such that a micromolar affinity aptamer common to small molecules can be detected at levels, orders of magnitude lower than the dissociation constant. The following selection protocol was used to isolate abers specific to cortisol. Dialysis and enrichment monitoring were used to show increased elution of the target molecule after later selection steps.

A decrease in elution was observed when less target molecule and longer capture probes were used in the final steps. Next generation sequencing was used to analyze the eluded DNA after several different rounds. Early on after the sixth round of selection, a low percentage of the sequences had increased copy number, which was ranked into 20 bins after 13 rounds.

A much larger percentage of the abers were in high copy number. Then by making the selection process more stringent. In rounds 14 and 15, nearly half the selected abers were in the first rank of copy number frequency.

There were about 3000 unique sequences in 30, 000 runs at this point. The selected AP was incubated with gold nanoparticles followed by addition of target or for comparison control substrates. This assay showed that the AP had more specificity to cortisol than colic acid or to While attempting this procedure.

It's important to control the stringency of selection in each round as well as to optimize the number of PCR cycles. After watching this video, you should have a good understanding of how to select a structure switching AP and perform a gold nanoparticle detection assay.