Biomedical subjects

G A Soukup

Publications and source records attributed to G A Soukup.

13 recordsLinked to original sources

PubMed2001

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.

Base Sequence↗

PubMed2001

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.

Allosteric Regulation↗

PubMed2000

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.

Allosteric Regulation↗

PubMed2000

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.

Allosteric Regulation↗

PubMed1999

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.

Allosteric Regulation↗

PubMed1999

Engineering precision RNA molecular switches.

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

Allosteric Regulation↗

PubMed1999

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.

Allosteric Regulation↗

PubMed1999

Relationship between internucleotide linkage geometry and the stability of RNA.

The inherent chemical instability of RNA under physiological conditions is primarily due to the spontaneous cleavage of phosphodiester linkages via intramolecular transesterification reactions. Although the protonation state of the nucleophilic 2'-hydroxyl group is a critical determinant of the rate of RNA cleavage, the precise geometry of the chemical groups that comprise each internucleotide linkage also has a significant impact on cleavage activity. Specifically, transesterification is expected to be proportional to the relative in-line character of the linkage. We have examined the rates of spontaneous cleavage of various RNAs for which the secondary and tertiary structures have previously been modeled using either NMR or X-ray crystallographic data. Rate constants determined for the spontaneous cleavage of different RNA linkages vary by almost 10,000-fold, most likely reflecting the contribution that secondary and tertiary structures make towards the overall chemical stability of RNA. Moreover, a correlation is observed between RNA cleavage rate and the relative in-line fitness of each internucleotide linkage. One linkage located within an ATP-binding RNA aptamer is predicted to adopt most closely the ideal conformation for in-line attack. This linkage has a rate constant for transesterification that is approximately 12-fold greater than is observed for an unconstrained linkage and was found to be the most labile among a total of 136 different sites examined. The implications of this relationship for the chemical stability of RNA and for the mechanisms of nucleases and ribozymes are discussed.

Adenosine Triphosphate↗

PubMed1999

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.

Acids↗

PubMed1999

Allosteric ribozymes sensitive to the second messengers cAMP and cGMP.

We have engineered allosteric ribozymes by combining modular rational design with combinatorial strategies. This new procedure was used to create allosteric ribozymes that are activated by specific nucleoside 3',5'-cyclic monophosphates (cNMPs). A random-sequence domain was attached to stem II of hammerhead ribozymes via a communication module that serves as an interface between ribozyme and the effector binding site. Subjecting this initial random pool to in vitro selection methods produced populations that respond, or cleave, only in the presence of specific effector molecules. From generation 18, 20 and 23, cGMP, cCMP and cAMP-specific responsive ribozymes, respectively, were isolated and characterized. These methods show great promise for engineering allosteric ribozymes and for creating new ligand-specific aptamers.

Allosteric Regulation↗

PubMed1998

Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli.

Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors. Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes. While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking. In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA. An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose. Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E.coli by disrupting transcriptional interference. Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo , despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro . Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.

Base Sequence↗

PubMed1996

Selection of RNAs that bind to duplex DNA at neutral pH.

RNA that are capable of binding duplex DNA in a site-specific manner have potential applications in gene therapy strategies. Such RNAs might be targeted to DNA sequences in a gene promoter and prevent initiation of transcription by occluding transcription factors and/or RNA polymerases. RNA oligonucleotides that bind homopurine/homopyrimidine DNA sequences by forming triple-helical complexes involving T.A.T and C+.G.C base-triplets can be rationally designed. However, the formation of such pyrimidine motif triple helices typically requires mildly acidic conditions. In addition, the proper oligonucleotide sequence must be optimally presented within a longer RNA transcript if it is to be synthesized in vivo. To address these issues, RNAs were selected from pools of random sequences for binding to a homopurine/homopyrimidine DNA sequence. RNAs selected for binding the duplex DNA target between pH 6.5 and pH 7.4 were characterized by sequence analysis and binding studies. All RNAs isolated by selection and amplification were found to contain a pyrimidine recognition sequence for binding the duplex DNA target via conventional triple helix formation. The selected approximately 85 nt RNAs have dissociation constants that approach, but do not surpass, the binding affinity of a 21 nt RNA oligonucleotide that binds the DNA target sequence by forming a canonical triple helix. The presence of a pyrimidine recognition sequence within a longer RNA transcript is not sufficient for high affinity. Experimental data and secondary structure predictions suggest that the context of the pyrimidine recognition sequence within selected RNAs is a very important determinant of DNA binding affinity. These studies provide insight into the development of RNA transcripts that may function as gene-specific repressors by forming triple helices with DNA in vivo.

Base Sequence↗

PubMedDate not supplied

Preparation of oligonucleotide-biotin conjugates with cleavable linkers.

A procedure is presented for preparing an oligonucleotide-biotin conjugate that is chemically cleavable through the reduction of a disulfide bond within the linker. Conjugation involves reaction of a primary amine with an N-hydroxysulfosuccinimide ester linked to biotin. The oligonucleotide can be liberated from streptavidin agarose containing immobilized conjugate under mild conditions (neutral pH, 50 mM dithiothreitol). This cleavable conjugate is useful for affinity purification applications.

Base Sequence↗