Insights into DNA catalysis from structural and functional studies of the 8-17 DNAzyme.

DNAzymes (deoxyribozymes) are single-stranded DNA molecules endowed with catalytic activity, obtained by in vitro selection. In the past 25 years, dozens of DNAzymes have been identified and employed for applicative purposes, yet our knowledge of the structural and mechanistic basis of DNA catalysis remains very limited. The RNA-cleaving 8-17 DNAzyme, which depends on divalent metal ions for function, is possibly the most studied catalytic DNA in terms of mechanism. It is very efficient, implying that it adopts a combination of distinct catalytic strategies, but until recently it was uncertain which strategies are at play and how they are implemented. Recently, however, new functional studies and the attainment of high-resolution X-ray structures of an 8-17 construct, have offered a great opportunity for a more detailed understanding of its mechanism. This review examines the functional information gathered on 8-17, in the light of the available crystal structures, pointing out the congruences and possible inconsistencies between the functional and structural data. We will analyze separately three aspects of the DNAzyme function: the structural requirements for catalysis, the role of metal ions and the influence of pH on activity. Ultimately, we will contrast the experimental data with a model for the 8-17 mechanism proposed in the crystallographic study, whereby one specific G residue (G14) acts as a general base and a metal-coordinated water molecule acts as a general acid. Throughout this analysis we will signal the most outstanding mechanistic issues that remain to be addressed, with implications for the broader field of DNA catalysis.

Spherical Nucleic Acid Enzyme (SNAzyme) Boosted Chemiluminescence miRNA Imaging Using a Smartphone.

As acute myocardial infarction (AMI) has now become a severe death threat to humans and may abruptly occur at home or outdoors where sophisticated equipment is not available, it is of great importance to develop facile methodologies for the point-of-care (POC) diagnosis of AMI. Toward this goal, here we build a sensing platform for chemiluminescence (CL) microRNA (miRNA) imaging with a smartphone as the portable detector, and for the first time we achieve visualization of AMI-related miRNAs in real patients' serum. We first construct a spherical nucleic acid enzyme (termed SNAzyme) derived from a dense layer of G-quadruplex (G4) DNAzyme formed on the gold nanoparticle core, which displays ∼100-fold and higher catalytic activity and improved resistance to nuclease degradation in a real blood sample as compared to those of the G4 DNAzyme itself. These unique features endow the SNAzyme-boosted CL platform with superior imaging performance for analyzing an AMI-related miRNA, miRNA-133a. This miRNA is employed to trigger the target-catalyzed hairpin assembly to produce a sticky dsDNA linker that captures the SNAzyme nanolabel onto the substrate. In this way, miRNA-133a is successfully detected, with a limit of detection of 0.3 pM (S/N = 3) and a high selectivity over other miRNA analogs in patients' blood. Given its unique features in physiological environments, our SNAzyme-boosted imaging platform holds great promise for use in the POC diagnosis of AMI.

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme.

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100mM K+, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. 1D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.

Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.

Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.

The 10-23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage.

The 10-23 DNA enzyme (10-23 DNAzyme), a single-stranded DNA (ssDNA) molecule, can efficiently and specifically cleave almost any target RNA molecules. Therefore, it is regarded as one of the promising tools in gene therapy. However, there are still some obstacles, such as low efficiency of cellular uptake and instability in vivo, in its application. Taking advantage of the mechanism of Moloney mouse leukemia virus (MMLV) reverse transcriptase (RT), we investigate the construction of a novel ssDNA expression vector in this study. In order to improve the expression efficiency, the mmlv-rt gene and ODN-PMT (an oligodeoxynucleotide including other essential sequences for generating ssDNA) were cloned into a single plasmid under the control of 2 separated promoters. The ability of the vector to generate specific 10-23 DNAzyme in mammalian cell was tested by constructing a tryptophan-aspartate-containing coat protein (taco) gene-specific 10-23 DNAzyme expression plasmid. The potential of the expressed 10-23 DNAzyme to suppress TACO expression was also investigated. Our results indicated that this vector generates desired 10-23 DNAzyme in mammalian cells. The expressed 10-23 DNAzyme targeting taco gene can reduce TACO expression both at mRNA level (by 78.26%) and at protein level (by 75.30%).

Hairpin DNAzymes: a new tool for efficient cellular gene silencing.

BACKGROUND: RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity. METHODS: We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against 32P-labelled cRNA encoding the muscle acetylcholine receptor (AChR) alpha-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced 125I-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR. RESULTS: We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species. CONCLUSION: hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications.