DNA-catalyzed hydrolysis of esters and aromatic amides.

We previously reported that DNA catalysts (deoxyribozymes) can hydrolyze DNA phosphodiester linkages, but DNA-catalyzed amide bond hydrolysis has been elusive. Here we used in vitro selection to identify DNA catalysts that hydrolyze ester linkages as well as DNA catalysts that hydrolyze aromatic amides, for which the leaving group is an aniline moiety. The aromatic amide-hydrolyzing deoxyribozymes were examined using linear free energy relationship analysis. The hydrolysis reaction is unaffected by substituents on the aromatic ring (ρ ≈ 0), suggesting general acid-catalyzed elimination as the likely rate-determining step of the addition-elimination hydrolysis mechanism. These findings establish that DNA has the catalytic ability to achieve hydrolysis of esters and aromatic amides as carbonyl-based substrates, and they suggest a mechanism-based approach to achieve DNA-catalyzed aliphatic amide hydrolysis.

DNA-catalyzed sequence-specific hydrolysis of DNA.

Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and with a rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is noteworthy because many natural DNA nucleases are bimetallic protein enzymes.

Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme.

We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5'-phosphate and 3'-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs=2.7 h(−1) and rate enhancement of 10(12) over the uncatalyzed P−O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson−Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson−Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson−Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its "recognition site" from ATGT (as for 10MD5) to ATGTT or larger, i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.

Systematic evaluation of the dependence of deoxyribozyme catalysis on random region length.

Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N(20) through N(60)) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N(40) regions. In the case of DNA cleavage, here we found that shorter N(20) and N(30) regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via ß-elimination, whereas longer N(50) and N(60) regions did not lead to catalytically active DNA sequences. Follow-up selections with N(20), N(30), and N(40) regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N(30) and N(60) regions provided catalytically active sequences, whereas N(20) was unsuccessful, and the N(40) deoxyribozymes were functionally superior (in terms of rate and yield) to N(30) and N(60). Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable.

New branched DNA constructs.

The Watson-Crick base pairing of DNA is an advantageous phenomenon that can be exploited when using DNA as a scaffold for directed self-organization of nanometer-sized objects. Several reports have appeared in the literature that describe the generation of branched DNA (bDNA) with variable numbers of arms that self-assembles into predesigned architectures. These bDNA units are generated by using cleverly designed rigid crossover DNA molecules. Alternatively, bDNA can be generated by using synthetic branch points derived from either nucleoside or non-nucleoside building blocks. Branched DNA has scarcely been explored for use in nanotechnology or from self-assembling perspectives. Herein, we wish to report our results for the synthesis, characterization, and assembling properties of asymmetrical bDNA molecules that are able to generate linear and circular bDNA constructs. Our strategy for the generation of bDNA is based on a branching point that makes use of a novel protecting-group strategy. The bDNA units were generated by means of automated DNA synthesis methods and were used to generate novel objects by employing chemical and biological techniques. The entities generated might be useful building blocks for DNA-based nanobiotechnology.