Biomedical subjects

N G Walter

Publications and source records attributed to N G Walter.

13 recordsLinked to original sources

PubMed1998

On-line polymerase chain reaction (PCR) monitoring.

In this short note, we present the results of a case study for monitoring the whole polymerase chain reaction (PCR) process (all steps) with a glass fiber fluorometer that was described in a former publication. To utilize this fluorometer, which was originally constructed for a PCR machine with three thermostating devices, a new thermostating device has been developed: the glass fiber matrix is integrated into the thermostating device, while the PCR samples are heated and cooled. The device is able to monitor all samples throughout all stages of PCR with the help of an intercalating dye. This approach also permits one to choose arbitrarily different cooling and heating rates.

Fluorometry↗

PubMed1998

The solvent-protected core of the hairpin ribozyme-substrate complex.

The complex between the hairpin ribozyme and its substrate consists of two domains that must interact in order to form a catalytic complex, yet experimental evidence concerning the points of interaction between the two domains has been lacking. Here, we report the use of hydroxyl radical footprinting to define the interface between the two domains. Cations that support very efficient ribozyme catalysis (magnesium and cobalt(III) hexammine) lead to the formation of a docked complex that features several regions of protection, indicating a solvent-inaccessible core within the tertiary structure of the complex. Cations that are suboptimal in cleavage reactions do not produce complexes with regions of reduced solvent accessibility. Nucleotides encompassing the substrate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected, suggesting their internalization into the catalytic core. Four distinct segments of the ribozyme are protected, including G11-A14, C25-C27, A38, and U42-A43. Protection of these sites is eliminated when g+1, an essential base at the cleavage site, is replaced by A. In addition, mutations which are known to decrease the fraction of docked complexes decrease or eliminate formation of a solvent-inaccessible core. Taken together, these observations demonstrate that we have identified the catalytic core of the active hairpin ribozyme-substrate complex.

Base Sequence↗

PubMed1998

Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme.

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

Genetic Engineering↗

PubMed1998

Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer.

The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min-1) but not for cleavage, where docking (0.5 min-1) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G+1A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.

Base Composition↗

PubMed1998

The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone.

BACKGROUND: The catalytic activity of RNA enzymes is thought to require divalent metal ions, which are believed to facilitate RNA folding and to play a direct chemical role in the reaction. RESULTS: We have found that the hammerhead, hairpin and VS ribozymes do not require divalent metal ions, their mimics such as [Co(NH3)6]3+, or even monovalent metal ions for efficient self-cleavage. The HDV ribozyme, however, does appear to require divalent metal ions for self-cleavage. For the hammerhead, hairpin and VS ribozymes, very high concentrations of monovalent cations support RNA-cleavage rates similar to or exceeding those observed in standard concentrations of Mg2+. Analysis of all reaction components by inductively coupled plasma-optical emission spectrophotometry (ICPOES) and the use of a variety of chelating agents effectively eliminate the possibility of contaminating divalent and trivalent metal ions in the reactions. For the hairpin ribozyme, fluorescence resonance energy transfer experiments demonstrate that high concentrations of monovalent cations support folding into the catalytically proficient tertiary structure. CONCLUSIONS: These results directly demonstrate that metal ions are not obligatory chemical participants in the reactions catalysed by the hammerhead, hairpin, and VS ribozymes. They permit us to suggest that the folded structure of the RNA itself contributes more to the catalytic function than was previously recognised, and that the presence of a relatively dense positive charge, rather than divalent metal ions, is the general fundamental requirement. Whether this charge is required for catalysis per se or simply for RNA folding remains to be determined.

Catalysis↗

PubMed1998

The hairpin ribozyme: structure, assembly and catalysis.

Recent studies of the hairpin ribozyme have revealed a distinct catalytic mechanism for this small RNA motif. Inner-sphere coordinated metal ions are not required, as the inert metal ion complex cobalt hexammine promotes catalysis. Detailed kinetic analyses have defined rates of individual steps in the catalytic cycle. Functional group modification, NMR studies of subdomains and cross-linking experiments, in combination with computer modeling, have led to a proposal for domain interactions in the substrate-ribozyme complex.

Models, Molecular↗

PubMed1997

Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small RNAs by recognizing pyrimidine residues.

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qbeta was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5'-d(TTTTTCC) was 5'-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qbeta replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 degrees C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qbeta replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qbeta replicase does not require a 2' hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A.T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qbeta replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows detection of RNA amplification by Qbeta replicase in real-time.

Base Sequence↗

PubMed1997

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems.

Fluorescein↗

PubMed1996

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR.

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe's average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 micrograms unspecific DNA without post-PCR probe manipulations could be achieved with different primer/ probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Automation↗

PubMed1996

Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis.

Binding kinetics in solution of six N,N,N',N'-tetramethyl-5-carboxyrhodamine-labeled oligodeoxyribonucleotide probes to a 101mer target RNA comprising the primer binding site for HIV-1 reverse transcriptase were characterized using fluorescence correlation spectroscopy (FCS). FCS allows a sensitive, non-radioactive real time observation of hybridization of probes to the RNA target in the buffer of choice without separation of free and bound probe. The binding process could directly be monitored by the change in translational diffusion time of the 17mer to 37mer DNA probe upon specific hybridization with the larger RNA target. The characteristic diffusion time through a laser-illuminated open volume element with 0.5 micron in diameter increased from 0.13-0.2 ms (free) to 0.37-0.50 ms (bound), depending on the probe. Hybridization was approximated by biphasic irreversible second-order reaction kinetics, yielding first-phase association rate constants between 3 x 10(4) and 1.5 x 10(6) M-1 s-1 for the different probes. These varying initial rates reflected the secondary structures of probes and target sites, being consistent with a hypothetical binding pathway starting from loop-loop interactions in a kissing complex, and completion of hybridization requiring an additional interaction involving single-stranded regions of both probe and target. FCS thus permits rapid screening for suitable antisense nucleic acids directed against an important target like HIV-1 RNA with low consumption of probes and target.

Animals↗

PubMed1995

Modelling viral evolution in vitro using exo- Klenow polymerase: continuous selection of strand displacement amplified DNA that binds an oligodeoxynucleotide to form a triple-helix.

Evolution comprises cycles of amplification, mutagenesis and selection. To study evolutionary phenomena, isothermal strand displacement amplification (SDA) of double-stranded DNA as an in vitro model for rolling-circle replication of viruses has been coupled to a positive selection procedure. First, two subsequent amplification reactions utilizing exo- Klenow polymerase were performed under direct observation using the fluorescent dye thiazole orange. Under the chosen conditions, the mutation rate was 1.5 x 10(-3) and 0.4 x 10(-3) for base substitutions and deletions, respectively. Then, a 16mer oligodeoxynucleotide with an acridine moiety coupled to its 5' end was used to select for double strands that retained their ability to form a triple-helix with the oligodeoxynucleotide. Conditions for triple-helix formation were chosen such that only 10 to 40% of the SDA products were allowed to bind the third strand. Non-denaturing polyacrylamide gel electrophoresis was used to separate triple-helices from unmodified double strands, and only triplex strands were used to initiate a new round of error-prone amplification and selection. Nine such rounds with about 270 molecular generations were performed. The final mutant spectrum was characterized and compared with those of amplification reactions without additional selection pressure. While without selection pressure base substitutions and deletions throughout the initial wild-type rapidly produce a diverse mutant distribution, the consensus after nine selection rounds clearly shows two mutational hotspot positions. Using gel shift assays and a newly developed non-radioactive DNase I footprinting technique, it could be shown that both the initial wild-type and the final consensus do not differ significantly in their triplex formation ability. As opposed to this, they do show different amplification efficiencies. The final consensus sequence is amplified with the highest rate in the exponential reaction phase, while the most abundant clone, which is characterized by two additional point deletions, is the sequence with the highest amplification rate in the linear growth phase.

Base Sequence↗

PubMed1995

Multichannel PCR and serial transfer machine as a future tool in evolutionary biotechnology.

As an improved strategy for producing functional macromolecules by in vitro evolutionary optimization, we propose an automated machine that can process up to 960 samples in parallel. It consists of a 960-well PCR machine with special sealed plastic reaction vessels and appropriate handling devices. We show that the heat-sealing technique does not significantly affect the activity of Taq DNA Polymerase or that of the temperature-sensitive Q beta RNA polymerase but avoids cross-contamination and evaporation of the samples. Initial experiments demonstrate the suitability of the apparatus to uniformly process the samples and to perform the thermocycling. Serial transfer of reaction products into fresh reaction solution was used to initiate further rounds of amplification as a typical experimental setup for an evolutionary biotechnology application.

Allolevivirus↗

PubMed1994

Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer.

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency.

Base Composition↗