New insight on the response of bacteria to fluoride.

Fluoride has been used for decades to prevent caries and it is well established that this anion can inhibit the growth of bacteria. However, the precise effects that fluoride has on bacteria and the mechanisms that bacteria use to overcome fluoride toxicity have largely remained unexplored. Recently, my laboratory reported the discovery of biological systems that bacteria use to sense fluoride and reduce fluoride toxicity. These sensors and their associated genes are very widespread in biology, which has implications for a number of issues that are central to the use of fluoride for dental care. Below I provide a summary of our findings, comment on some of the key prospects for expanding our understanding of fluoride's effects on biology, and propose some future uses of this knowledge.

Improved genetic transformation methods for the model alkaliphile Bacillus halodurans C-125.

AIMS: Bacillus halodurans C-125 is a Gram-positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C-125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. METHODS AND RESULTS: A plasmid artificial modification system (PAM system, Yasui et al. 2008) for B. halodurans C-125 was created that increases transformation efficiency by 10- to 1000-fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. CONCLUSIONS: Use of the B. halodurans C-125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C-125. SIGNIFICANCE AND IMPACT OF THE STUDY: These techniques allow routine genetic manipulation of B. halodurans C-125, a model alkaliphilic bacterium with important industrial properties.

Engineering ligand-responsive gene-control elements: lessons learned from natural riboswitches.

In the last two decades, remarkable advances have been made in the development of technologies used to engineer new aptamers and ribozymes. This has encouraged interest among researchers who seek to create new types of gene-control systems that can be made to respond specifically to small-molecule signals. Validation of the fact that RNA molecules can exhibit the characteristics needed to serve as precision genetic switches has come from the discovery of numerous classes of natural ligand-sensing RNAs called riboswitches. Although a great deal of progress has been made toward engineering useful designer riboswitches, considerable advances are needed before the performance characteristics of these RNAs match those of protein systems that have been co-opted to regulate gene expression. In this review, we will evaluate the potential for engineered RNAs to regulate gene expression and lay out possible paths to designer riboswitches based on currently available technologies. Furthermore, we will discuss some technical advances that would empower RNA engineers who seek to make routine the production of designer riboswitches that can function in eukaryotes.

Allosteric nucleic acid catalysts.

Endowing nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA as controllable therapeutic agents or as sensors of their cognate effector compounds. The ability to engineer RNA catalysts that are allosterically regulated by effector binding has been propelled by the union of modular rational design principles and powerful combinatorial strategies.

Deoxyribozymes: new players in the ancient game of biocatalysis.

The repetitive and extraordinarily stable polynucleotide chains of DNA serve as an ideal storage system for genetic information. Although it is best known for its helical structure and relatively inert character, in vitro selection can be used to compel DNA to perform a surprising variety of chemical reactions. These artificial DNA enzymes or 'deoxyribozymes' generate large chemical rate enhancements and demonstrate precise substrate recognition, much like their protein and RNA counterparts. Recent studies with these prototypic deoxyribozymes indicate that DNA has a substantial untapped potential for intricate structure formation that could be exploited in novel chemical and biological catalysis.

DNA enzymes.

Biological catalysis is dominated by enzymes that are made of protein, but several distinct classes of catalytic RNAs are known to promote chemical transformations that are fundamental to cellular metabolism. Is biological catalysis limited only to these two biopolymers, or is DNA also capable of functioning as an enzyme in nature? To date, no DNA enzymes of natural origin have been found. However, an increasing number of catalytic DNAs, with characteristics that are similar to those of ribozymes, are being produced outside the confines of the cell. An assessment of the potential for structure formation by DNA leads to the conclusion that DNA might have considerable latent potential for enzymatic function.

Immobilized RNA switches for the analysis of complex chemical and biological mixtures.

A prototype biosensor array has been assembled from engineered RNA molecular switches that undergo ribozyme-mediated self-cleavage when triggered by specific effectors. Each type of switch is prepared with a 5'-thiotriphosphate moiety that permits immobilization on gold to form individually addressable pixels. The ribozymes comprising each pixel become active only when presented with their corresponding effector, such that each type of switch serves as a specific analyte sensor. An addressed array created with seven different RNA switches was used to report the status of targets in complex mixtures containing metal ion, enzyme cofactor, metabolite, and drug analytes. The RNA switch array also was used to determine the phenotypes of Escherichia coli strains for adenylate cyclase function by detecting naturally produced 3',5'- cyclic adenosine monophosphate (cAMP) in bacterial culture media.

Cooperative binding of effectors by an allosteric ribozyme.

An allosteric ribozyme that requires two different effectors to induce catalysis was created using modular rational design. This ribozyme construct comprises five conjoined RNA modules that operate in concert as an obligate FMN- and theophylline-dependent molecular switch. When both effectors are present, this 'binary' RNA switch self-cleaves with a rate enhancement of approximately 300-fold over the rate observed in the absence of effectors. Kinetic and structural studies implicate a switching mechanism wherein FMN binding induces formation of the active ribozyme conformation. However, the binding site for FMN is rendered inactive unless theophylline first binds to its corresponding site and reorganizes the RNA structure. This example of cooperative binding between allosteric effectors reveals a level of structural and functional complexity for RNA that is similar to that observed with allosteric proteins.

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.

DNA aptamers and DNA enzymes.

Investigators using combinatorial methods are revealing the surprising structural and functional abilities of DNA. A consequence of DNA's structure-forming potential is its ability to form highly specific receptors and ligands, and even its ability to catalyze chemical reactions. Unlike the classical images of double-stranded DNA, these DNA structures have many of the higher-ordered structural features that are found with ribozymes and other folded RNAs. Recent research is beginning to indicate that these new DNA structures are not rare exceptions, and that DNA, despite the absence of 2' hydroxyl groups, could rival RNA in its ability to form intricate structures and in its ability to function as an enzyme.

Altering molecular recognition of RNA aptamers by allosteric selection.

In a continuing effort to explore structural and functional dynamics in RNA catalysis, we have created a series of allosteric hammerhead ribozymes that are activated by theophylline. Representative ribozymes exhibit greater than 3000-fold activation upon effector-binding and cleave with maximum rate constants that are equivalent to the unmodified hammerhead ribozyme. In addition, we have evolved a variant allosteric ribozyme that exhibits an effector specificity change from theophylline to 3-methylxanthine. Molecular discrimination between the two effectors appears to be mediated by subtle conformational differences that originate from displacement of the phosphodiester backbone near the effector binding pocket. These findings reveal the importance of abstruse aspects of molecular recognition by nucleic acids that are likely to be unapproachable by current methods of rational design.

Rational design of allosteric ribozymes.

BACKGROUND: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes. RESULTS: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created. CONCLUSIONS: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation-a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.

A DNA enzyme with Mg(2+)-dependent RNA phosphoesterase activity.

BACKGROUND: Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approximately 10(-4) min-1 in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 degrees C. The Mg(2+)-dependent reaction is more difficult, with an uncatalyzed rate of approximately 10(-7) min-1 under comparable conditions. Mg(2+)-dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+)-dependent reaction. RESULTS: We generated a population of > 10(13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg2+, Mn2+, Zn2+ or Pb2+. Examination of individual clones from the Mg2+ lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction. This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01 min-1. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. CONCLUSIONS: We have generated a Mg(2+)-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approximately 10(5)-fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

A DNA enzyme that cleaves RNA.

BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

In vitro selection of self-cleaving DNAs.

BACKGROUND: Ribozymes catalyze an important set of chemical transformations in metabolism, and 'engineered' ribozymes have been made that catalyze a variety of additional reactions. The possibility that catalytic DNAs or 'deoxyribozymes' can be made has only recently been addressed. Specifically, it is unclear whether the absence of the 2' hydroxyl renders DNA incapable of exhibiting efficient enzyme-like activity, making it impossible to discover natural or create artificial DNA biocatalysts. RESULTS: We report the isolation by in vitro selection of two distinct classes of self-cleaving DNAs from a pool of random-sequence oligonucleotides. Individual catalysts from 'class I' require both Cu2+ and ascorbate to mediate oxidative self-cleavage. Individual catalysts from class II use Cu2+ as the sole cofactor. Further optimization of a class II individual by in vitro selection yielded new catalytic DNAs that facilitate Cu2+-dependent self-cleavage with rate enhancements exceeding 1 000 000-fold relative to the uncatalyzed rate of DNA cleavage. CONCLUSIONS: Despite the absence of 2' hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.

Are engineered proteins getting competition from RNA?

Progress in several areas of research is pushing back the supposed limitations of nucleic acid structure and function. New ligand-binding and catalytic RNAs are being created at a rapid pace. Some engineered RNAs offer potential as therapeutic agents whereas others can be used as model systems to study the principles that direct structure formation, molecular recognition and catalytic function by nucleic acids.

Inventing and improving ribozyme function: rational design versus iterative selection methods.

Two major strategies for generating novel biological catalysts exist. One relies on our knowledge of biopolymer structure and function to aid in the 'rational design' of new enzymes. The other, often called 'irrational design', aims to generate new catalysts, in the absence of detailed physicochemical knowledge, by using selection methods to search a library of molecules for functional variants. Both strategies have been applied, with considerable success, to the remodeling of existing ribozymes and the development of ribozymes with novel catalytic function. The two strategies are by no means mutually exclusive, and are best applied in a complementary fashion to obtain ribozymes with the desired catalytic properties.

Nucleic acid molecular switches.

Natural and artificial ribozymes can catalyse a diverse range of chemical reactions. Through recent efforts in enzyme engineering, it has become possible to tailor the activity of ribozymes to respond allosterically to specific effector compounds. These allosteric ribozymes function as effector-dependent molecular switches that could find application as novel genetic-control elements, biosensor components or precision switches for use in nanotechnology.