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.

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.

Covalent tagging of phosphorylated peptides by phosphate-specific deoxyribozymes.

[Image: see text] Phosphorylated tyrosine and serine residues in peptides are modified selectively by DNA catalysts (see the figure). The deoxyribozymes catalyze covalent attachment of an RNA tag to a range of peptide sequences, establishing proof-of-principle for a new approach to phosphopeptide analysis.

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.

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.

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.