The biggest breakthrough our lab made in the field of hybridization probes was made in 2007, by Dmitry Kolpashchikov, who suggested an ADF splitting hybridization probes in halves, assessing each of the half, its own function, for example, increasing selectivity to mismatches and single nucleotide variations, along to unwinding complex probes. The second breakthrough was made a decade later when it was suggested to add additional multi-component binding arms along with the uniting platform. Such designs were able to work even with complex targets, for example, double-stranded nucleic acids and highly-structured nucleic acids.
The biggest challenges our lab faces right now are coming from the problem of low sensitivity of DNA nano sensors in comparison to conventional amplification techniques. By now, the DNA nano sensors in amplification free assay presents low selectivity, and we are trying to overcome this problem with new molecular designs and new ways of DNA nano sensors detection. The main advantages of our DNA sensors are their sensitivity in terms of flow detected concentration of the analog and selectivity.
For example, their ability to detect single nucleotide polymorphism. Also as an advantage compared to other diagnostics techniques, our DNA sensors are able to unwind and detect complex RNA structures and double stranded DNA. The new scientific question our results have pathed the way forward is, is it possible to bind double stranded DNA using protein-free hybridization props?
The protein independent prop could be easily accessible by automated DNA synthesis, easier to be delivered in cells and be compatible with chemical modification and complex nanostructures developed by DNA nanotechnology. Combined with DNAzymes, such as DNA-cleaving DNAzymes. Such a prop could become a basis for protein-free nucleases, a useful tool for gene editing and therapy.
To begin, open the UNAFold web tool and insert the selected genomic region of interest. Modify the folding temperature to 55 degrees Celsius, the concentration of monovalent ions to 250 millimolars and the magnesium ion concentration to 200 millimolars. Next, open the resulting secondary structure of the single stranded DNA fragment and select a stable hairpin structure.
Locate the DNA nano machines binding site either in unstructured regions or on the loops of the hairpin structures for enhanced binding efficiency. If the target analyte site exhibits a stem loop structure, place arm two on one side of the stem covering the loop and three to five nucleotides on the second stem side. Position arm one on a fragment of the second stem side.
Now, open the DINAMelt tool and examine the sequences of both the arms and their reverse complement sequences. Combine arms one and two sequences with the DNAzyme cores halves, and the F-sub binding fragments. Next, construct a random sequence for the DNA tile fragment, ensuring it is not shorter than the length of arm three.
Combine this sequence with the arm three sequence using a linker. Connect arm two to a sequence that is complimentary to the DNA tile fragment via a suitable linker. At last, using the UNA fold web app, analyze the secondary structure of the newly designed DNA strands.
To begin, design DNAzyme-based nano machines or DNM with a 10 to 23 core using free software. Obtain the designed sequences from commercial sources. Then take 0.5 milliliter micro centrifuge tubes with 200 microliters of the reaction buffer.
Add DZB tile and tile arm three oligonucleotides to the tubes. Gently mix them and spin down the solution. Wrap the lid of the closed tube with paraffin film or use screw cap tubes.
Incubate the tube in a 500 milliliter beaker with boiling water for two minutes. Then turn the heater off and allow the temperature to passively cool down overnight. Now prepare the 12%native page using the given reagents.
Transfer the mixture to the gel cassette and allow it to polymerize. Mix one microliter of each sample with one microliter of four times loading dye, and load them into the gel. Place the cassette into the chamber.
Fill it with TBE buffer and run the gel for 90 minutes at 80 to 100 volts. Dye the gel with athydium bromide, and visualize it using a gel documenting system. To begin, design DNA-based nano machines or DNM with a 10 to 23 core using free software.
Assemble the DNM and check its size and homogeneity by native page. Then prepare 160 microliters of F-sub in the reaction buffer. Prepare seven aliquots of 160 microliters of preassembled DNM F-sub and Dza in the reaction buffer.
Add the analyte to tubes two to seven and gently mix the components. Then spin down the tubes and divide the solutions into three 50 microliter portions in a black 96-well plate. Seal the plate with an optically transparent film.
Incubate the plate in a water bath at 55 degrees Celsius for one hour. Then spin down the plate. Afterward, measure fluorescence at 480 nanometers excitation wavelength, and 525 nanometers emission wavelength.
