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.

In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification.

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.

Riboswitches in eubacteria sense the second messenger cyclic di-GMP.

Cyclic di-guanosine monophosphate (di-GMP) is a circular RNA dinucleotide that functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes, including cell differentiation, conversion between motile and biofilm lifestyles, and virulence gene expression. However, the mechanisms by which cyclic di-GMP regulates gene expression have remained a mystery. We found that cyclic di-GMP in many bacterial species is sensed by a riboswitch class in messenger RNA that controls the expression of genes involved in numerous fundamental cellular processes. A variety of cyclic di-GMP regulons are revealed, including some riboswitches associated with virulence gene expression, pilus formation, and flagellum biosynthesis. In addition, sequences matching the consensus for cyclic di-GMP riboswitches are present in the genome of a bacteriophage.

Deoxyribozymes: new activities and new applications.

DNA in its single-stranded form has the ability to fold into complex three-dimensional structures that serve as highly specific receptors or catalysts. Only protein enzymes and ribozymes are known to be responsible for biological catalysis, but deoxyribozymes with kinetic parameters that rival ribozymes can be created in the laboratory. Some of these engineered DNA catalysts are showing surprising potential as therapeutic agents, which makes them biologically relevant if not biologically derived. If DNA's natural role is strictly genomic, how significant is its innate catalytic prowess? New examples of engineered deoxyribozymes serve as empirical examples of the potential for catalysis by DNA. These results indicate that the true catalytic power of DNA is limited by discovery and not by chemistry.

Characterization of a DNA-cleaving deoxyribozyme.

A copper-dependent self-cleaving DNA that was isolated by in vitro selection has been minimized to its smallest active domain using both in vitro selection and rational design methods. The minimized 46-nucleotide deoxyribozyme forms duplex and triplex substructures that flank a highly conserved catalytic core. This self-cleaving construct can be converted into a bimolecular complex that comprises separate substrate and enzyme domains. Substrate cleavage is directed at one of two adjacent nucleotides and proceeds via an oxidative cleavage mechanism that is unique to the position cleaved. The structural, kinetic and mechanistic characteristics of this DNA-cleaving deoxyribozyme are reported.

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.

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.

In vitro selection of kinase and ligase deoxyribozymes.

Exploration of the limits of biocatalysis has led to the discovery that DNA has significant potential for enzymatic function. This makes possible the construction of DNA enzymes or "deoxyribozymes" for catalyzing various chemical reactions that could be used to address fundamental questions in biocatalysis or that could find unique applications in biotechnology. Of significant interest are self-modification reactions, given the fundamental role that DNA serves in modern living systems. Recently, in vitro selection strategies have been used to isolate prototypical ATP-dependent deoxyribozymes from random-sequence populations of DNA that catalyze DNA phosphorylation and others that catalyze DNA adenylation. In nature, protein enzymes such as T4 DNA kinase and T4 DNA ligase catalyze identical chemical reactions. These findings suggest that DNA constructs could be engineered to efficiently catalyze other self-modifying reactions, including ATP-dependent DNA ligation. This article provides a detailed overview of the methods used to isolate deoxyribozymes that promote ATP-dependent DNA ligation.

Selection for catalytic function with nucleic acids.

For in vitro selection of catalytic polynucleotides, each new protocol must be designed to harness the desired catalytic activity to help propel the selection process itself. This unit gives guidelines for design of in vitro selection experiments for catalytic function. It outlines several representative protocols as examples of successful selection experiments, providing a conceptual basis for the design and implementation of new selective-amplification protocols for nucleic acids.

Molecular recognition of cAMP by an RNA aptamer.

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.

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.

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.

Structural diversity of self-cleaving ribozymes.

In vitro selection was used to isolate Mg(2+)-dependent self-cleaving ribozymes from random sequence. Characterization of representative clones revealed the emergence of at least 12 classes of ribozymes that adopt distinct secondary structure motifs. Only one class corresponds to a previously known structural motif, that of the naturally occurring hammerhead ribozyme. Each ribozyme promotes self-cleavage via an internal phosphoester transfer reaction involving the adjacent 2'-hydroxyl group with a chemical rate enhancement of between 10(3)- and 10(6)-fold greater than the corresponding uncatalyzed rate. These findings indicate that RNA can form a multitude of secondary and tertiary structures that promote cleavage by internal phosphoester transfer. Upon further in vitro selection, a class I ribozyme that adopts an "X motif" structure dominates over all other ribozymes in the population. Thus, self-cleaving RNAs isolated by in vitro selection from random-sequence populations can rival the catalytic efficiency of natural ribozymes.

Capping DNA with DNA.

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.

Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP.

RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3',5'-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.