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

31 조회수. 시작하려면 무료 소프트웨어를 사용하여 10-23 코어로 DNAzyme 기반 나노 머신 (DNM)을 설계하십시오. 상업적 출처에서 설계된 시퀀스를 얻습니다. 그런 다음 0.5 밀리리터 마이크로 원심분리기 튜브와 200 마이크로리터의 반응 완충액을 취합니다.DZB-Tile 및 Tile-arm3 올리고뉴클레오티드를 튜브에 추가합니다. 부드럽게 섞고 용액을 회전시킵니다. 닫힌 ...

비디오: 기능적 특성화를 위한 DNAzyme-Based Nanomachine의 조립

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Designing DNAzyme-Based Nanomachines with a 10-23 Core for Detection of an Epstein-Barr Virus Fragment

To begin, open the UNA fold 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.

Limit of Detection (LOD) Assay to Check the Sensitivity of DNAzyme-Based Nanomachines (DNM)

To begin, design DNAzyme-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.