Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

We are developing new types of fluorescent hybridization probes for sequence-specific nucleic acid analysis. Most recently, hybridization analysis tools have expanded with assays that take advantage of CRISPR-Cas systems. Currently, state-of-the-art hybridization probes, such as TaqMan, and molecular beacon probes, are most used to analyze nucleic acid targets.

Accurate detection of single nucleotide substitutions and nucleic acid targets is still practically challenging. Our protocol ensures the required level of cell activity while providing a label-free fluorescent signal read out. To begin, design the sequences of two unmodified DNA oligonucleotide strands constituting SLAS.

Use a fragment containing nucleotides for 6 to 39 of DAP-10-42. Convert stem 1 consisting of nucleotides 1 to 8 and 36 to 42 into stem 1 prime by removing a bulging thymine at position 5, shortening the stem to four base pairs and adding a terminal cytosine-guanine base pair. Include nucleotides 9 to 29 in one SLAS strand and nucleotides 30 to 35 in the other.

Extend the three prime terminal sequence of the fragment with nucleotides 9 to 29 by adding deoxy GGTCAT. Then extend the five prime terminal sequence of the 30 to 35 fragment with deoxy TGACC to form a six-base pair stem 2. Now extend stem 1 prime on both strands with deoxy TT linkers, and DNA sequences complementary to the nucleic acid target.

This yields the sequences of strands SLAS-U and SLAS-S constituting the probe. Make SLAS-S complimentary to a 7 to 10 nucleotide region. That includes the single nucleotide substitution site.

Then ensure that the target binding arm of SLAS-U is complimentary to a 15 to 25 nucleotide fragment adjacent to the SLAS-S region. Assess the melting temperatures of the target binding duplexes using the UNAFold web server. Click on the INAMelt tab.

Go to Applications and select Two State Melting Hybridization. Enter the interacting sequences in five prime to three prime order in the left and right boxes. Then adjust the assay temperature to 22 degrees Celsius and input the monovalent and divalent cation concentrations to 20 millimolar and 25 millimolar ions, respectively.

Adjust the concentrations of the interacting sequences in the corresponding box. Press submit and review the Gibbs energy change, enthalpY, entropy, and melting temperature values for the corresponding duplexes. Confirm that the melting temperatures for perfectly matched targets and the target binding arms of SLAS-S and SLAS-U are above the assay temperature, 22 degrees Celsius.

Ensure that the duplex between SLAS-S and a mismatched target yields melting temperatures below the assay temperature to maintain specificity. If necessary, adjust the lengths of target binding arms to meet these conditions. Obtain the finalized SLAS-U and SLAS-S oligonucleotide strands from a commercial DNA supplier or synthesize them in-house using an automated DNA synthesizer.

Prepare stock solutions of Auramine O, SLAS-S, SLAS-U, and assay buffer. Prepare the master mix containing all assay components but the target. Add nuclease-free water to the final five by sixth volume of the master mix.

Then vortex and spin the master mix. Dispense 50 microliters into each sample tube. Next, label one sample as a no target blank and one as a positive control.

Add 10 microliters of target-containing sample to a tube containing the master mix to make a 60-microliter sample. For the blank, add 10 microliters of nuclease-free water. Then pipette 10 microliters of synthetic DNA oligonucleotide containing the target sequence into the positive control tube.

Mix all samples in centrifuge briefly using a microcentrifuge. Then incubate the tubes at 22 degrees Celsius for 10 to 60 minutes. Measure fluorescence at 540 nanometers upon excitation at 475 nanometers using a fluorescent spectrophotometer.

SLAS was tailored to target a specific fragment of the NANOGP8 gene. Target M was fully complimentary to the SLAS-S strand, while target MM had a cytosine nucleotide position 1, 423, introducing a mismatch with SLAS-S. Upon addition of the fully complimentary target M, fluorescence increased steadily and plateaued after 45 to 50 minutes.

However, a clear signal was detectable within 10 minutes with a signal-to-blank ratio of 10 SLAS showed high fluorescence signal for fully matched targets, but not for mismatched targets or blanks. Fluorescence output increased linearly with target concentration up to 500 nanomolar, enabling quantification and determination of detection limits. PCR amplified samples showed varying levels of signal with only Sample 2 exceeding the fluorescence threshold value of 2.

Based on the calibration curve, the concentration of NANOGP8 amplicon in Sample 2 was estimated at 124+or 13 nanomolar. Fluorescence detection of SLAS signal was consistent across both a benchtop spectrophotometer and a portable fluorometer. A signal-to-blank ratio above 20 was also visually observed using UV light.