Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn2+ and Mn2+ cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (44) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N40 DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA

Dynamic allosteric control of noncovalent DNA catalysis reactions

Allosteric modulation of catalysis kinetics is prevalent in proteins and has been rationally designed for ribozymes. Here, we present an allosteric DNA molecule that, in its active configuration, catalyzes a noncovalent DNA reaction. The catalytic activity is designed to be modulated by the relative concentrations of two DNA regulator molecules, one an inhibitor and the other an activator. Dynamic control of the catalysis rate is experimentally demonstrated via three cycles of up and down regulation by a factor of over 10. Unlike previous works, both the allosteric receptor and catalytic core are designed, rather than evolved. This allows flexibility in the sequence design and modularity in synthetic network construction

Operation of a DNA-Based Autocatalytic Network in Serum

The potential for inferring the presence of cancer by the detection of miRNA in human blood has motivated research into the design and operation of DNA-based chemical amplifiers that can operate in bodily fluids. As a first step toward this goal, we have tested the operation of a DNA-based autocatalytic network in human serum and mouse serum. With the addition of sodium dodecyl sulfate to prevent degradation by nuclease activity, the network was found to operate successfully with both DNA and RNA catalysts

Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, 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.biochem.5b00691A trivalent lanthanide (Ln3+)-dependent RNA-cleaving DNAzyme, Ce13d, was recently isolated via in vitro selection. Ce13d is active in the presence of all Ln3+ ions. Via introduction of a single phosphorothioate (PS) modification at the cleavage site, its activity with Ln3+ decreases while all thiophilic metals can activate this DNAzyme. This property is unique to Ce13d and is not found in many other tested DNAzymes. This suggests the presence of a well-defined but general metal binding site. Herein, a systematic study of Ce13d with the PO substrate (using Ce3+) and the PS substrate (using Cd2+) is performed. In both the PO and PS systems, the highest activity was with ∼10 μM metal ions. Higher concentrations of Ce3+ completely inhibit the activity, while Cd2+ only slows the activity. A comparison of different metal ions suggests that the role of metal is to neutralize the phosphate negative charge. Both systems follow a similar pH–rate profile with a single deprotonation step, indicating similar reaction mechanisms. The activity difference between the Rp and Sp form of the PS substrate is <10-fold, which is much smaller than most known RNA-cleaving enzymes. Mutation studies identified eight highly conserved purines, among which the two adenines play mainly structural roles, while the guanines are likely to be involved in metal binding. Ce13d can serve as a model system for further understanding of DNAzyme biochemistry and bioinorganic chemistry.Natural Sciences and Engineering Research Council || Grant number: 386326

DNA hybridization catalysts and catalyst circuits

Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that carry out their functions through the catalysis of metastable fuels into waste products. Catalytic control of reaction rates will prove to be as useful and ubiquitous in DNA nanotechnology as it is in biology. Here we present experimental results on the control of the decay rates of a metastable DNA "fuel". We show that the fuel complex can be induced to decay with a rate about 1600 times faster than it would decay spontaneously. The original DNA hybridization catalyst [15] achieved a maximal speed-up of roughly 30. The fuel complex discussed here can therefore serve as the basic ingredient for an improved DNA hybridization catalyst. As an example application for DNA hybridization catalysts, we propose a method for implementing arbitrary digital logic circuits

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

In Vitro Selection of Mercury (II)- and Arsenic (V)-Dependent RNA-Cleaving DNAzymes

Abstract DNAzymes (or catalytic DNA) are cell-free biomolecular recognition tools with target recognition sequences for charged molecules such as metal ions, antibiotics, and pharmaceuticals. In this study, using in vitro selection, large populations (e.g., 1015) of random DNA sequences were used as the raw material for the selection of “catalytic or functional molecules” for Hg2+ and As5+. From a random pool of 45-nt (Pool-A) and 35-nt (Pool-B) templates, we isolated RNA-cleaving catalytic Hg2+- and As5+- active DNAzymes, respectively. After eight cycles of selection and amplification wihin Pool A, sequences were enriched with a 54% cleavage efficiency against Hg2+. Similarly, Pool B was found to catalyze ca. 18% cleavage efficiency against As5+ after 10 cycles of repeated selection and amplification. The M-fold software analysis resulted in sequences in the two active pools being dominated by AATTCCGTAGGTCCAGTG and ATCTCCTCCTGTTC functional motifs for Hg2+- and As5+-based catalysis, respectively. These DNAzymes were found to have higher activity in the presence of transition metal ions compared to alkaline earth metal ions. A maximum cleavage rate of 2.7 min−1 for Hg2+ was found to be highest in our study at a saturating concentration of 500 μM. Results demonstrate that DNAzymes are capable of selectively binding transition metal ions, and catalytic rates are at par with most Mg2+-dependent nucleic acid enzymes under similar conditions, and indicate their potential as metal species-specific biosensors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63396/1/ees.2006.0026.pd

DNA catalysts as phosphatases and as phosphoserine lyases

Proteins and RNA are the only known biopolymers that have catalytic roles in nature, whereas DNA is primarily considered to store and transfer genetic information. However, artificial single-stranded DNA has been identified by in vitro selection to catalyze several chemical reactions and several of those are of biological relevance. For in vitro selection or directed evolution of proteins, direct amplification is not possible, and it essential to attach the genotype to the phenotype. For nucleic acids however, the functional biopolymer can be readily amplified. DNA has the advantage of being directly amplified by polymerases, whereas RNA requires an additional reverse transcription step. Moreover, DNA catalysts identified by in vitro selection processes have shown similar catalytic proficiency as RNA. DNA has added advantages of low cost of chemical synthesis and higher stability. Considering these factors combined, identification of artificial DNA catalysts (deoxyribozymes) for chemical reactions is a valuable endeavor with long-term implications. Protein post-translational modifications (PTMs) are highly important in biological processes involving cellular regulation. Additionally, PTMs serve as important intermediates or key motifs on natural products and bioactive peptides. The natural protein enzymes carrying out the essential modifications may have several shortcomings for biotechnological use. Identification of artificial DNA catalysts with ability to perform chemoselective post-translation chemical reaction would be highly useful in studying biological regulatory processes, performing synthesis and late-stage diversification of post-translationally modified peptides, as well as carrying out other important functions that natural proteins may not readily solve. My first effort was to identify DNA enzymes with peptide/protein phosphatase activity, more specifically dephosphorylation of peptide side chains. Without a catalyst, phosphomonoester hydrolysis reactions have exceedingly low spontaneous reaction rates. Nature utilizes proficient protein enzymes to perform this challenging reaction with great efficiency. Using a known DNA catalyst for the in vitro selection process, new DNA catalysts were identified with phosphatase activity. The phosphatase DNA catalysts exhibited multiple-turnover activity with phosphotyrosine-containing free peptides and were active even in the presence of externally added cell lysate or bovine serum albumin (BSA). Furthermore, the best DNA phosphatase functioned with a larger protein substrate. This established the fundamental ability of DNA to catalyze dephosphorylation of amino acid side chain residues. The study also suggested that phosphatase DNA catalysts could perform intracellular phosphatase activity. Hence, these deoxyribozymes were functionalized on gold nanoparticles and delivered inside live mammalian cells to investigate if they behave as functional protein analogues (or mimics) of recombinantly expressed Protein Tyrosine Phosphatase (PTP1B). Separately, efforts were directed towards the important goal of identifying sequence-selective phosphatase deoxyribozymes. Although three separate efforts were directed towards identifying sequence-selective phosphatases deoxyribozymes, we were unsuccessful in accomplishing this specific goal of selectivity in the context of peptide sequence discrimination. Dehydroalanine (Dha) is a non-proteinogenic electrophilic amino acid that serves as a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides and microcystins. DNA enzymes were identified to establish the fundamental catalytic ability to eliminate phosphate from phosphoserine (pSer) to form Dha, namely phosphoserine lyase activity. Furthermore, DhaDz1 was utilized to achieve chemo-enzymatic synthesis of a cyclic cystathionine-containing peptide. Based on this initial success, future efforts will be directed to achieve sequence-general phosphoserine and phosphotyrosine lyase activity. Separately, application of sequence-general lyases in the synthesis of complex lanthipeptides and enrichment of phosphopeptides/proteins in phosphoproteomics will be explored

In Vitro Selection of Hg (II) and As (V)-Dependent RNA-Cleaving DNAzymes

DNAzymes (or catalytic DNA) are cell-free biomolecular recognition tools with target recognition sequences for charged molecules such as metal ions, antibiotics, and pharmaceuticals. In this study, using in vitro selection, large populations (e.g., 1015) of random DNA sequences were used as the raw material for the selection of "catalytic or functional molecules" for Hg2+ and As5+. From a random pool of 45-nt (Pool-A) and 35-nt (Pool-B) templates, we isolated RNA-cleaving catalytic Hg2+- and As5+-active DNAzymes, respectively. After eight cycles of selection and amplification wihin Pool A, sequences were enriched with a 54% cleavage efficiency against Hg2+. Similarly, Pool-B was found to catalyze ca. 18% cleavage efficiency against As5+ after 10 cycles of repeated selection and amplification. The M-fold software analysis resulted in sequences in the two active pools being dominated by "AATTCCGTAGGTCCAGTG" and "ATCTCCTCCTGTTC" functional motifs for Hg2+- and As5+-based catalysis, respectively. These DNAzymes were found to have higher activity in the presence of transition metal ions compared to alkaline earth metal ions. A maximum cleavage rate of 2.7 min−1 for Hg2+ was found to be highest in our study at a saturating concentration of 500 µM. The results demonstrate that DNAzymes are capable of selectively binding transition metal ions, and catalytic rates are at par with most Mg2+-dependent nucleic acid enzymes under similar conditions, and indicate their potential as metal-species specific biosensors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63104/1/ees.2007.24.73.pd