New DNA-hydrolyzing DNAs isolated from an ssDNA library carrying a terminal hybridization stem.

DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by self-hydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of ∼20 conserved nucleotides and a half-life of ∼15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors.

MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches.

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.

A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype.

Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k(obs), maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.

Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection.

In vitro selection has been used extensively over the past 10 years to create functionally diverse DNA enzymes. The majority of in vitro selection experiments to date have focused on the outcome rather than the process itself, a process that remains to be fully elucidated. In vitro selection techniques rely on the probability that some DNA molecules in a random-sequence library will fold into an appropriate tertiary structure and catalyze a desired reaction. Thus, sufficient sequence diversity in the DNA pool (and hence more catalytic DNA sequences) is a prerequisite for the successful isolation of efficient deoxyribozymes. The catalytic sequence diversity established by in vitro selection is governed largely by the choice of selection pressures, one of which is the length of the reaction time. The objective of this study was to evaluate the sequence diversity change of a pool of RNA-cleaving deoxyribozymes as a function of the reaction time. Seventeen rounds of in vitro selection were performed, and the reaction time was progressively decreased from 5 h to 5 s. A representative population from each time class was subsequently cloned and sequenced. A decline in sequence diversity was observed with decreasing reaction time, and the relationship appears to be logarithmic. In contrast, a control selection performed with a constant reaction time during each round led to a linear and comparatively very slow decrease in sequence diversity. This study provides the first methodical examination of the change in catalytic sequence diversity that occurs through the course of a deoxyribozyme selection experiment. Moreover, it represents a first step toward fully understanding the intricate pathway that lies between the beginning and end of an in vitro selection experiment.

Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme.

We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme--an RNA-cleaving catalytic DNA motif previously reported--from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans, and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine tuning when different dinucleotide junctions are given as cleavage sites.

Characterization of a DNA-cleaving deoxyribozyme.

A copper-dependent self-cleaving DNA that was isolated by in vitro selection has been minimized to its smallest active domain using both in vitro selection and rational design methods. The minimized 46-nucleotide deoxyribozyme forms duplex and triplex substructures that flank a highly conserved catalytic core. This self-cleaving construct can be converted into a bimolecular complex that comprises separate substrate and enzyme domains. Substrate cleavage is directed at one of two adjacent nucleotides and proceeds via an oxidative cleavage mechanism that is unique to the position cleaved. The structural, kinetic and mechanistic characteristics of this DNA-cleaving deoxyribozyme are reported.