assembly of dnazyme-based nanomachines for its functional characterization

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

One of the earliest methods for&#160;nucleic acid detection is&#160;complementary binding of selective oligonucleotides to the analyzed RNA or DNA sequences. This method employs nucleic acid fragments (probes) that can form Watson-Crick base pairs with a targeted DNA or RNA analyte1. The complex is then differentiated from the analyte and the unbound probe by a variety of techniques. Certain methods, such as Northern blotting, in situ hybridization, or qPCR, are based on the&#160;phenomenon&#160;of complementary binding2. The most common hybridization probes have two significant drawbacks: low sensitivity (they can reach micromolar to nanomolar levels) as well as low selectivity to single nucleotide variations (SNV), including point mutations. The issue requires a sophisticated approach since the solutions to these drawbacks conflict with each other3. To provide the best selectivity, the detecting oligonucleotide should be kept short enough to form unstable hybrids with mismatched analytes. Such short oligonucleotides are not able to bind targeted nucleic acids tightly and unwind their secondary structures, thus often failing to provide a detectable signal. It is especially challenging to detect CG-rich fragments in biological RNA, or double-stranded DNA (dsDNA). On the other hand, long probes are insensitive to SNV3. To break the deadlock, the concept of a binary probe has been&#160;developed4.
Binary sensors split a single detection probe into two pieces and specialize its fragments, keeping one of them long to unwind the hairpins or invade the dsDNA. The second binary probe fragment remains short to be sensitive to a mismatched base, thus providing a means for detecting SNV. The signal will be produced if the two binary sensor parts are joined together4. The full complex can be visualized via FRET5, molecular beacon probe6,7, or G-quadruplexes with peroxidase-like activity8,9,10, or else RNA-cleaving11,12,13 DNAzyme. Binary sensors with a 10-23 DNAzyme core have been amply described. They have been adapted to detect single-stranded nucleic acid fragments&#160;created synthetically11, or single-stranded amplification products14,15. Detection without amplification is also possible, for example, in the case of 16S rRNA extracted from a cell culture, since this molecule is abundant in cells12,16. Binary sensors with a 10-23 DNAzyme core can also be adapted for non-nucleic acid detection, such as for lead ions17. Nevertheless, low sensitivity is still considered to be one of the major drawbacks of binary 10-23 DNAzyme sensors, and&#160;various approaches, including cascades18 or alternative signal-enhancing methods19, have been developed to address this issue. Unfortunately, the above techniques drastically increase the cost and instability of the sensor components, and so they have not come into practice.
The experiment described in this work uses 10-23 DNAzyme-based DNA-nanosensors referred to as DNA-nanomachines (DNM)20,21. These structures include binary sensors and also&#160;(1) DNA facilitators flanking the binary probe,&#160;unwinding the structured regions and&#160;invading into double-stranded DNAs, and (2) a DNA tile that holds all the DNM parts together in close proximity and increases the chances of signal formation (Figure 1). Altogether, the 10-23 DNAzyme-based DNMs decrease the limit of detection (LOD) down to the picomolar range21; they can be used to detect 16rRNA in cell lysates22 or report the presence of viral RNA without amplification23,24. At the same time, the DNMs remain sensitive to the SNV and can even be used to genotype alleles in heterozygous organisms25. The DNM method can be used as a complementary technique to any amplification type for product visualization or identification of the product in a complex mixture with high selectivity. The DNMs, unlike conventional TaqMan and beacon probes, do not require heating of the sample and so can be used in end-point detection protocols, making the overall detection cost-effective.
This article describes the in silico development of a DNM and demonstrates procedures for assembly and functional characterization of DNM. The work was performed using a synthetic fragment of Epstein-Barr virus (EBV) corresponding to the positions 13972-14154 of the viral DNA (OR652423.1) (see Table of materials).

Author Spotlight: Advancements in DNA Nanosensors &amp;#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

The biggest breakthrough our lab made in the field of hybridization probe was made in 2007 by Dmitry Kolpashchikov, who suggested an ADL 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 Unitan 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 sensor detection.The main advantages of our DNA sensors are their sensitivity in terms of low detected concentration of the analyte 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 paved the way for is, is it possible to find 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 nano structures developed by DNA nanotechnology combined with DNA enzymes such as Dnase lipids and DNA enzymes. Such a prop could become a basis for protein free nucleases a useful tool for gene editing and therapy.

Assembly of DNAzyme-Based Nanomachines for its Functional Characterization

To begin, design DNAzyme-based nanomachines, or DNM, with a 10 to 23 core, using free software. Obtain the designed sequences from commercial sources. Then, take 0.5 milliliter microcentrifuge tubes with 200 microliters of the reaction buffer.Add DZB-Tile and Tile-arm3 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 82 to 100 volts. Dye the gel with ethidium bromide and visualize it using a gel documenting system.

assembly-dnazyme-based-nanomachines-for-its-functional

Research

JoVE Journal

Biochemistry

20 Views. To begin, design DNAzyme-based nanomachines, or DNM, with a 10 to 23 core, using free software. Obtain the designed sequences from commercial sources. Then, take 0.5 milliliter microcentrifuge tubes with 200 microliters of the reaction buffer.Add DZB-Tile and Tile-arm3 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 ...