A deoxyribozyme that hydrolyzes DNA phosphodiester linkages with a requirement for both Zn2+ and Mn2+ is switched by only two nucleotide mutations to require Zn2+ alone, demonstrating that DNA-catalyzed DNA hydrolysis can be achieved using only one metal ion cofactor.

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 (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. 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.

A homogeneous fluorescence-based DNA detection system has been developed to measure DNA in protein solutions. The technique relies on the increase in fluorescence of a dye molecule when it intercalates into double-stranded (ds) DNA. The increased fluorescence is a direct measurement of the amount of DNA in the sample. The analysis time required per sample is less than 5 min. The dye has absorbance and emission maxima at 485 and 530 nm, respectively. The assay is linear from 98 pg/mL to 200 ng/mL of DNA in buffers containing no proteins with typical relative standard deviation values of less than 2.4%. The assay performance was evaluated under various matrix conditions, including buffers, pH, ionic salts, detergents, denaturants and organic solvents. Each reagent was tested at several concentrations to determine how the slope and linearity (r value) of the standard curve were affected. Even in the presence of matrix components and protein, the assay was able to quantitatively detect picogram to nanogram levels of DNA. The fluorescence can be removed by DNase treatment. This method is specific for dsDNA with RNA emitting less than 2% intensity of an equivalent mass of DNA.