Engineered Allosteric Ribozymes That Sense the Bacterial Second Messenger Cyclic Diguanosyl 5′-Monophosphate

A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-5′-monophosphate (c-di-GMP) have been created by using in vitro selection. An RNA library was generated by using random-sequence bridges to join a hammerhead self-cleaving ribozyme to an aptamer from a natural c-di-GMP riboswitch. Specific bridge sequences, called communication modules, emerged through two in vitro selection efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptamer. Representative RNAs were found that exhibit EC₅₀ (half-maximal effective concentration) values for c-di-GMP as low as 90 nM and IC₅₀ (half-maximal inhibitory concentration) values as low as 180 nM. The allosteric RNAs display molecular recognition characteristics that mimic the high discriminatory ability of the natural aptamer. Some engineered RNAs operate with ribozyme rate constants approaching that of the parent hammerhead ribozyme. By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in three mutant strains of Escherichia coli were quantitatively estimated from cell lysates. Our findings demonstrate that engineered c-di-GMP-sensing ribozymes can be used as convenient tools to monitor c-di-GMP levels from complex biological or chemical samples. Moreover, these ribozymes could be employed in high-throughput screens to identify compounds that trigger c-di-GMP riboswitch function

Retraining and Optimizing DNA-Hydrolyzing Deoxyribozymes for Robust Single- and Multiple-Turnover Activities

Recently, we reported two classes of Zn²⁺-dependent DNA-hydrolyzing deoxyribozymes. The class I deoxyribozymes can adopt a secondary structure of either hairpin or stem-loop-stem. The corresponding most active representatives, I-R1 and I-R3, exhibit single-turnover <i>k</i>_obs values of ∼0.059 and ∼1.0 min^–1 at 37 °C, respectively. Further analysis revealed that I-R3 could perform slow multiple-turnover catalysis with a <i>k</i>_cat of ∼0.017 min^–1 at 37 °C. In this study, we sought to retrain and optimize the class I deoxyribozymes for robust single- and multiple-turnover cleavage activities. Refined consensus sequences were derived based on the data of <i>in vitro</i> reselection from the degenerate DNA pools. By examining individual candidates, we obtained the I-R1 mutants I-R1a-c with improved single-turnover <i>k</i>_obs values of 0.68–0.76 min^–1 at 37 °C, over 10 times faster than I-R1. Meanwhile, we further demonstrated that I-R1a–c and I-R3 are thermophilic. As temperature went higher beyond 45 °C, I-R3 cleaved faster with the <i>k</i>_obs value reaching its maximum of ∼3.5 min^–1 at 54 °C. Using a series of the <i>k</i>_obs values of I-R3 from 37 to 54 °C, we calculated the apparent activation energy <i>E</i>_a to be ∼15 ± 3 kcal/mol for the DNA-catalyzed hydrolysis of DNA phosphodiester bond. In addition, we were able to design a simple yet efficient thermal-cycling protocol to boost the effective <i>k</i>_cat of I-R3 from 0.017 to 0.50 min^–1, which corresponds to an ∼30-fold improvement of the multiple-turnover activity. The data and findings provide insights on the enzymatic robustness of DNA-catalyzed DNA hydrolysis and offer general strategies to study various DNA enzymes

Small, Highly Active DNAs That Hydrolyze DNA

DNA phosphoester bonds are exceedingly resistant to hydrolysis in the absence of chemical or enzymatic catalysts. This property is particularly important for organisms with large genomes, as resistance to hydrolytic degradation permits the long-term storage of genetic information. Here we report the creation and analysis of two classes of engineered deoxyribozymes that selectively and rapidly hydrolyze DNA. Members of class I deoxyribozymes carry a catalytic core composed of only 15 conserved nucleotides and attain an observed rate constant (<i>k</i>_obs) of ∼1 min^–1 when incubated near neutral pH in the presence of Zn²⁺. Natural DNA sequences conforming to the class I consensus sequence and structure were found that undergo hydrolysis under selection conditions (2 mM Zn²⁺, pH 7), which demonstrates that the inherent structure of certain DNA regions might promote catalytic reactions, leading to genomic instability

Identification of Ligand Analogues that Control c-di-GMP Riboswitches

Riboswitches for the bacterial second messenger c-di-GMP control the expression of genes involved in numerous cellular processes such as virulence, competence, biofilm formation, and flagella synthesis. Therefore, the two known c-di-GMP riboswitch classes represent promising targets for developing novel modulators of bacterial physiology. Here, we examine the binding characteristics of circular and linear c-di-GMP analogues for representatives of both class I and II c-di-GMP riboswitches derived from the pathogenic bacterium <i>Vibrio choleae</i> (class I) and <i>Clostridium difficile</i> (class II). Some compounds exhibit values for apparent dissociation constant (<i>K</i>_D) below 1 μM and associate with riboswitch RNAs during transcription with a speed that is sufficient to influence riboswitch function. These findings are consistent with the published structural models for these riboswitches and suggest that large modifications at various positions on the ligand can be made to create novel compounds that target c-di-GMP riboswitches. Moreover, we demonstrate the potential of an engineered allosteric ribozyme for the rapid screening of chemical libraries for compounds that bind c-di-GMP riboswitches