Part:BBa_K4933004

DNAzyme FBS2

DNAzymes are engineered with three recognition loops (RecLoops). The secondary structure of this DNAzyme looks like a ‘T’ shaped molecule with loops on all ends. The upper arms contain a region that is hidden due to strand overlap
When two specific and complimentary sequences bind to the RecLoop regions, this masking DNA strand is displaced (also known as strand displacement). This allows the masked region to be exposed and the substrate/probe (which is a complement to the hidden region) to hybridize with that region. This substrate has a fluorophore and quencher attached to either ends of it. Upon hybridization, the distance between the ends increase leading to a fluorescence signal.
A third loop is equipped with a deactivation sequence, granting us precise control over the DNAzyme's activity. This unique feature endows our platform with the capacity for logical decision-making. In practical terms, a positive signal is generated solely when two specific sequences (recSeq) are present. This allows us to differentiate between strains containing two recSeqs and strains containing all three recSeqs.
This basic part has its RecLoop sequence in such a way it detects for CoVID specific sequences and identify delta strain from the omicron strain

Usage and Biology

The T-stems of the DNAzyme is essentially double stranded which helped to keep the folded structure and maintain the loops at its ends. These loops exert a tension to open up. This tension is compensated by the base pairing in the stem region.
When sequences complementary to the loop regions are introduced, the tension is increased. Depending on the number of complimentary bindings in the loop regions, the tension will keep on increasing and after a certain threshold of total number of complimentary binding, the tension will exceed the T-loop binding strength, causing the fold to open up.
This threshold of required tension needed to unfold can be increased by increasing the GC content in T arm regions thus increasing the binding strength. The upper two arms of the T structure, when folded, masks the active site of the DNAzyme. When unfolded, this active site is exposed which then can bind to and cleave the fluorescence substrate resulting in the fluorescence signal.

Modelling

2D folding of DNAzyme
The 2D folded structure of the aptamer was generated using mFOLD (UNAFOLD web server). The input was the DNAzyme sequence and parameters 37°C, 0.01M Na⁺ and 0.1M Mg⁺⁺, linear DNA, oligomer correction, 5 percent suboptimality. The structure with least energy had the following values:
dG = -13.37 kcal/mol
dH = -187.00 kcal/mol
dS = -559.83kcal/mol-K
T_m = 60.9°C
The below image is the least energy (most stable structure)

3D structure of Aptamer
The 3D folded structure of the aptamer was generated using Xiao Lab 3dRNA/DNA - An RNA and DNA tertiary structure prediction method (http://biophy.hust.edu.cn/new/3dRNA/create).
To visualize, PyMOL software was use PyMOL:The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.
The input to Xiao Lab 3dRNA/DNA was the DNA aptamer sequence, the 2D structure in Vienna format, obtained from mFOLD and parameters molecule type DNA, Procedure Best, # of Predictions 5. No changes were made in Advanced options

DNAzyme in complete bound configuration

DNAzyme with single loop hybridized with complimentary target (given in white)

DNAzyme with both loop hybridized with complimentary targets (given in white). This is essentially the activated DNAzyme

DNAzyme with NOT loop hybridized with complimentary target (given in white). This is essentially the deactivated DNAzyme.

Molecular Dynamics Simulation
We Initiated our study by utilizing CHARMM-GUI to prepare and configure the DNAzyme system for the subsequent Molecular Dynamics (MD) simulation. Our simulation was conducted on NAMD on a High-Performance COmputing (HPC) platform, allowing us to explore the dynamic behavior of the DNAzyme molecule when it encounters its complementary probe.
We analyzed the Root-Mean-Squared Deviation values (RMSD) and Root-Mean-Square_Fluctuation (RMSF) which provided us with insights into the dynamic behavior of the system. The 3D visualization of the DNAzyme’s structure highlighting the intricate conformational changes it undergoes. These findings helped us in opening doors to a deeper understanding of the DNAzyme offering us a potential avenue for enhancement.
This research underscores the power of computational biology in resolving the mysteries of molecular dynamics and serves as a benchmark for further studies.

Characterization

We ordered the DNAzyme template as a double stranded sequence from Twist Bioscience. We performed two different asymmetric PCRs to generate two different ssDNA which effectively. One of the ssDNA will be produced by primers compliment to our actual DNAzyme sequence. The other ssDNA will be produced using the primers compliment to the DNAzyme sequence. The new DNAzyme, compliment to our reported DNAzyme, will act as a negative control for our systems.
Upon receiving we resuspended the DNA sample in 8 micro liters of Nuclease Free Water to get 100mM solution Then we took the nanodrop of the prepared solution

At the same time, we also ordered the single stranded DNA which acted as the template for our DNAzyme. For this the sample was resuspended in 1.161mL of Nuclease free water to get 100mM solution and took nanodrop of this solution.

From the prepared solutions, we performed asymmetric PCR to get amplified quantities of our DNAzyme
This was then followed with Gel Electrophoresis to get our DNAzyme at the 105bp position and then extracted using column filtration.
The purified, amplified obtained DNAzyme was then subjected to CD spectroscopy to correlate its structure with the simulated data. In CD spectroscopy, unbound regions show two valleys at 210nm and 280nm. Bound regions show single peak around 280nm.

Thus the presence of a valley at 210nm, no valley or peak at 280nm and combined with the molecular weight confirms production of DNAzyme by asymmetric PCR and its structure containing both unbound and bound regions
Finally the activated DNAzyme was mixed with the fluorescence substrate and the subsequent signal was recorded and analyzed.

References

[1] Genome Sequences of the Delta Variant (B.1.617.2) and the Kappa Variant (B.1.617.1) Detected in Morocco: https://doi.org/10.1128%2FMRA.00727-21 accession numbers MZ208926 and MZ571142
[2] SARS-CoV-2 B.1.1.529 (Omicron) Variant — United States, December 1–8, 2021: https://doi.org/10.15585%2Fmmwr.mm7050e1
[3] SARS-CoV-2 genome sequences: GISAID database identifiers: EPI_ISL_2110643 and EPI_ISL_2966236 NCBI GeneBank Accession numbers: MZ208926 and MZ571142
[4] Analysis Tool Web Services from the EMBL-EBI. (2013) McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, Cowley AP, Lopez R Nucleic acids research 2013 Jul;41(Web Server issue):W597-600 doi:10.1093/nar/gkt376
[5] Mfold web server for nucleic acid folding and hybridization prediction: https://doi.org/10.1093/nar/gkg595
[6] Vienna RNA downloadable package https://tbi.univie.ac.at/RNA/
[7] Medium Scale Integration of Molecular Logic Gates in an Automaton: https://doi.org/10.1021/nl0620684
[8] Automated and fast building of three-dimensional RNA structures: https://doi.org/10.1038/srep00734
[9] GROMACS: A message-passing parallel molecular dynamics implementation: https://doi.org/10.1016/0010-4655(95)00042-E
[10] Kalé, L.V., Bhatele, A., Bohm, E.J., Phillips, J.C. (2011). NAMD (NAnoscale Molecular Dynamics). In: Padua, D. (eds) Encyclopedia of Parallel Computing. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-09766-4_505
[11]SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data: https://doi.org/10.1101%2Fgr.138545.112
[12]PyMOL: The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.