Mitigation of bacterial adhesion and subsequent biofilm formation is quickly becoming a strategy for the prevention of hospital-acquired infections. We demonstrate a basic strategy for surface modification that combines the ability to control attachment by microbes with the ability to inactivate microbes. The surface consists of two active materials: poly(p-phenylene ethynylene)-based polymers, which can inactivate a wide range of microbes and pathogens, and poly(N-isopropylacrylamide)-based polymers, which can switch between an hydrophobic "capture" state and a hydrophilic "release" state. The combination of these materials creates a surface that can both bind microbes in a switchable way and kill surface-bound microbes efficiently. Considerable earlier work with cationic poly(p-phenylene ethynylene) polyelectrolytes has demonstrated and characterized their antimicrobial properties, including the ability to efficiently destroy or deactivate Gram-negative and Gram-positive bacteria, fungi, and viruses. Similarly, much work has shown (1) that surface-polymerized films of poly(N-isopropylacrylamide) are able to switch their surface thermodynamic properties from a swollen, relatively hydrophilic state at low temperature to a condensed, relatively hydrophobic state at higher temperature, and (2) that this switch can control the binding and/or release of microbes to poly(N-isopropylacrylamide) surfaces. The active surfaces described herein were fabricated by first creating a film of biocidal poly(p-phenylene ethynylene) using layer-by-layer methods, and then conferring switchable adhesion by growing poly(N-isopropylacrylamide) through the poly(p-phenylene ethynylene) layer, using surface-attached polymerization initiators. The resulting multifunctional, complex films were then characterized both physically and functionally. We demonstrate that such films kill and subsequently induce widespread release of Gram-negative and Gram-positive bacteria.

Pistol ribozyme (Psr) is a distinct class of small endonucleolytic ribozymes, which are important experimental systems for defining fundamental principles of RNA catalysis and designing valuable tools in biotechnology. High-resolution structures of Psr, extensive structure−function studies, and computation support a mechanism involving one or more catalytic guanosine nucleobases acting as a general base and divalent metal ion-bound water acting as an acid to catalyze RNA 2′-O-transphosphorylation. Yet, for a wide range of pH and metal ion concentrations, the rate of Psr catalysis is too fast to measure manually and the reaction steps that limit catalysis are not well understood. Here, we use stopped-flow fluorescence spectroscopy to evaluate Psr temperature dependence, solvent H/D isotope effects, and divalent metal ion affinity and specificity unconstrained by limitations due to fast kinetics. The results show that Psr catalysis is characterized by small apparent activation enthalpy and entropy changes and minimal transition state H/D fractionation, suggesting that one or more pre-equilibrium steps rather than chemistry is rate limiting. Quantitative analyses of divalent ion dependence confirm that metal aquo ion pK a correlates with higher rates of catalysis independent of differences in ion binding affinity. However, ambiguity regarding the rate-limiting step and similar correlation with related attributes such as ionic radius and hydration free energy complicate a definitive mechanistic interpretation. These new data provide a framework for further interrogation of Psr transition state stabilization and show how thermal instability, metal ion insolubility at optimal pH, and pre-equilibrium steps such as ion binding and folding limit the catalytic power of Psr suggesting potential strategies for further optimization.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross–Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a “plateau” in the pH–rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn /k 0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL–rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.

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