Merely two mutations switch a DNA-hydrolyzing deoxyribozyme from heterobimetallic (Zn/Mn) to monometallic (Zn-only) behaviorw

Metal ions can significantly expand the scope of macromolecular catalysis. For catalysts composed of nucleic acids (DNA or RNA), metal ions are typically required for both structural and catalytic roles. Divalent metal ions such as Mg, Mn, and Zn are often, but not always, obligatory cofactors for ribozymes and deoxyribozymes. We recently described the 10MD5 deoxyribozyme as an example of a highly active DNA catalyst that hydrolyzes DNA phosphodiester linkages of single-stranded DNA substrates. Because uncatalyzed P–O bond hydrolysis is extremely slow (t1/2 E 30 million years), 5 the hydrolysis achieved by 10MD5 (t1/2 E 15 min) represents a considerable 10 12 rate enhancement. 10MD5 was identified by in vitro selection in the presence of a mixture of Mg, Mn, and Zn; its activity requires both Mn and Zn. The Kd,app for Mn 2+ is B5 mM and the optimal Zn concentration is 1.0 mM, with significant loss of rate and yield at either lower or higher Zn concentrations. Another salient characteristic of 10MD5 is its rather sharp pH optimum. Catalysis is optimal near pH 7.5; pH changes of merely 0.2 units in either direction lead to a much lower DNA hydrolysis rate constant (at least six-fold decrease) and yield (at least two-fold decrease). We subsequently used in vitro evolution to identify a quintuple mutant of 10MD5, named 9NL27, that is more tolerant to a range of pH values. In the present study, we evaluated the divalent metal ion requirements of 10MD5, 9NL27, and related sequence variants. We found that only two nucleotide mutations in 10MD5 are sufficient to switch the metal ion dependence from heterobimetallic (Zn and Mn) to strictly monometallic (Zn only), with both practical and mechanistic implications. The 9NL27 deoxyribozyme is one of several 10MD5 variants that emerged upon in vitro evolution of 10MD5 for improved pH tolerance. 9NL27 and 10MD5 differ at only five out of 40 nucleotides in the originally random enzyme region, which is located between two fixed binding arm sequences