Reduced Retinal Microvascular Density, Improved Forepaw Reach, Comparative Microarray and Gene Set Enrichment Analysis with c-jun Targeting DNA Enzyme

Retinal neovascularization is a critical component in the pathogenesis of common ocular disorders that cause blindness, and treatment options are limited. We evaluated the therapeutic effect of a DNA enzyme targeting c-jun mRNA in mice with pre-existing retinal neovascularization. A single injection of Dz13 in a lipid formulation containing N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine inhibited c-Jun expression and reduced retinal microvascular density. The DNAzyme inhibited retinal microvascular density as effectively as VEGF-A antibodies. Comparative microarray and gene expression analysis determined that Dz13 suppressed not only c-jun but a range of growth factors and matrix-degrading enzymes. Dz13 in this formulation inhibited microvascular endothelial cell proliferation, migration and tubule formation in vitro. Moreover, animals treated with Dz13 sensed the top of the cage in a modified forepaw reach model, unlike mice given a DNAzyme with scrambled RNA-binding arms that did not affect c-Jun expression. These findings demonstrate reduction of microvascular density and improvement in forepaw reach in mice administered catalytic DNA.This work was supported by grants from Cancer Institute NSW and the National Health and Medical Research Council (NHMRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Faster Triphosphorylation Ribozyme.

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1

DNAzyme Hybridization, Cleavage, Degradation and Sensing in Undiluted Human Blood Serum

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acs.analchem.5b00220.RNA-cleaving DNAzymes provide a unique platform for developing biosensors. However, a majority of the work has been performed in clean buffer solutions, while the activity of some important DNAzymes in biological sample matrices is still under debate. Two RNA-cleaving DNAzymes (17E and 10-23) are the most widely used. In this work, we carefully studied a few key aspects of the 17E DNAzyme in human blood serum, including hybridization, cleavage activity, and degradation kinetics. Since direct fluorescence monitoring is difficult due to the opacity of serum, denaturing and nondenaturing gel electrophoresis were combined for studying the interaction between serum proteins and DNAzymes. The 17E DNAzyme retains its activity in 90% human blood serum with a cleavage rate of 0.04 min–1, which is similar to that in the PBS buffer (0.06 min–1) with a similar ionic strength. The activity in serum can be accelerated to 0.3 min–1 with an additional 10 mM Ca2+. As compared to 17E, the 10-23 DNAzyme produces negligible cleavage in serum. Degradation of both the substrate and the DNAzyme strand is very slow in serum, especially at room temperature. Degradation occurs mainly at the fluorophore label (linked to DNA via an amide bond) instead of the DNA phosphodiester bonds. Serum proteins can bind more tightly to the 17E DNAzyme complex than to the single-stranded substrate or enzyme. The 17E DNAzyme hybridizes extremely fast in serum. With this understanding, the detection of DNA using the 17E DNAzyme is demonstrated in serum.University of Waterloo || Natural Sciences and Engineering Research Council || Foundation for Shenghua Scholar of Central South University|| National Natural Science Foundation of China || Grant No. 21301195 Fellowship from the China Scholarship Council || CSC, Grant No. 20140637011

Searching for a DNAzyme version of the leadzyme

The final publication is available at Springer via http://dx.doi.org/10.1007/s00239-015-9702-zThe leadzyme refers to a small ribozyme that cleaves a RNA substrate in the presence of Pb2+. In an optimized form, the enzyme strand contains only two unpaired nucleotides. Most RNA-cleaving DNAzymes are much longer. Two classical Pb2+-dependent DNAzymes, 8–17 and GR5, both contain around 15 nucleotides in the enzyme loop. This is also the size of most RNA-cleaving DNAzymes that use other metal ions for their activity. Such large enzyme loops make spectroscopic characterization difficult and so far no high-resolution structural information is available for active DNAzymes. The goal of this work is to search for DNAzymes with smaller enzyme loops. A simple replacement of the ribonucleotides in the leadzyme by deoxyribonucleotides failed to produce an active enzyme. A Pb2+-dependent in vitro selection combined with deep sequencing was then performed. After sequence alignment and DNA folding, a new DNAzyme named PbE22 was identified, which contains only 5 nucleotides in the enzyme catalytic loop. The biochemical characteristics of PbE22 were compared with those of the leadzyme and the two classical Pb2+-dependent DNAzymes. The rate of PbE22 rises with increase in Pb2+ concentration, being 1.7 h−1 in the presence of 100 μM Pb2+ and reaching 3.5 h−1 at 500 µM Pb2+. The log of PbE22 rate rises linearly in a pH-dependent fashion (20 µM Pb2+) with a slope of 0.74. In addition, many other abundant sequences in the final library were studied. These sequences are quite varied in length and nucleotide composition, but some contain a few conserved nucleotides consistent with the GR5 structure. Interestingly, some sequences are active with Pb2+ but none of them were active with even 50 mM Mg2+, which is reminiscent of the difference between the GR5 and 8–17 DNAzymes.University of Waterloo || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Silver Nanoparticle Oligonucleotide Conjugate For Targeted Gene Silencing

This project explored a gene-regulated chemotherapy using a silver nanoparticle (SNP) conjugated with deoxyribozyme (DNAzyme) oligonucleotides which target a mutated gene in select cancer cells, sensitize them to doxorubicin treatment. Light exposure to the SNP-DNAzyme conjugates disengages the oligonucleotides and permits specific cleavage of the Kirsten Rat Sarcomal Oncogene Homolog (K-RAS) mRNA. These conjugates could provide spatiotemporal specificity in killing only those photoexposed cells with the mutant gene. Synthesis, functionalization and characterization of citrate and hydroxypropyl cellulose SNP conjugates confirmed attachment and photolytic release of the thiol-modified 10-23 DNAzyme. Gel electrophoresis was used to demonstrate DNAzyme photoactivation, showing greater K-RAS RNA degradation when disengaged compared to the SNP-tethered form. DNAzyme in the tethered form was also protected from DNAse degradation compared to photolyzed DNAzyme. Characterization of the toxicity and localization of the nanoparticle drug delivery system constructed for the release of a photolabile DNA oligonucleotide was checked within several sets of cells to check for temporal and spatial control efficiency. MTS, alamar blue, and flow cytometry assays were performed to assess cell viability in several sets of cell cultures, including HEK293 and MCF-7 (wildtype K-RAS), SW480 and MDA-MB-231 (mutant K-RAS), and 3T3 (negative control) lines. Following the 5-day experimental protocol involving staggered treatment with SNP-DNAzyme, UV light, and doxorubicin, no cell group showed the intended pattern of necrosis in mutant K-RAS cells without morbidity in controls or partial treatments. Thus further evaluation of K-RAS+/- cells which respond consistently in viability assays is necessary before this strategy can be deemed of potential as a targeted therapeutic

DNA Matrix Operation Based on the Mechanism of the DNAzyme Binding to Auxiliary Strands to Cleave the Substrate

Numerical computation is a focus of DNA computing, and matrix operations are among the most basic and frequently used operations in numerical computation. As an important computing tool, matrix operations are often used to deal with intensive computing tasks. During calculation, the speed and accuracy of matrix operations directly affect the performance of the entire computing system. Therefore, it is important to find a way to perform matrix calculations that can ensure the speed of calculations and improve the accuracy. This paper proposes a DNA matrix operation method based on the mechanism of the DNAzyme binding to auxiliary strands to cleave the substrate. In this mechanism, the DNAzyme binding substrate requires the connection of two auxiliary strands. Without any of the two auxiliary strands, the DNAzyme does not cleave the substrate. Based on this mechanism, the multiplication operation of two matrices is realized; the two types of auxiliary strands are used as elements of the two matrices, to participate in the operation, and then are combined with the DNAzyme to cut the substrate and output the result of the matrix operation. This research provides a new method of matrix operations and provides ideas for more complex computing systems