Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures.     

Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5' TpA 3' sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.     

Rapid hybridization kinetics of DNA attached to submicron latex particles.

We describe a novel method for attaching any DNA molecule to submicron latex beads and characterize the hybridization kinetic properties of these bead-DNA conjugates. The conjugates hybridize to DNA in solution with rates comparable to homogeneous hybridization reactions, are compatible with common hybridization conditions and are conveniently manipulated. They should thus serve as useful reagents for the fractionation and characterization of DNA and RNA.     

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.     

A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.     

Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support. This correlation shows a characteristic course for a particular coupling method. DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage. Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports. Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).     

A new method to monitor the rate of conformational transitions in RNA.

Many RNAs need Mg2+to produce stable tertiary structures. Here we describe a simple method to measure the rate and activation parameters of tertiary structure unfolding that exploits this Mg2+dependence. Our approach is based on mixing an RNA solution with excess EDTA in a stopped-flow instrument equipped with an absorbance detector, under conditions of temperature and ionic strength where, after chelation of Mg2+, tertiary structure unfolds. We have demonstrated the utility of this method by studying phenylalanine-specific transfer RNA from yeast (tRNAPhe) because the unfolding rates and the corresponding activation parameters have been determined previously and provide a benchmark for our technique. We find that within error, our stopped-flow method reproduces both the rate and activation enthalpy for tertiary unfolding of yeast tRNAPhe measured previously by temperature-jump relaxation kinetics. Since many different RNAs require divalent magnesium for tertiary structure stabilization, this technique should be applicable to study the folding of other RNAs.     

Measurement of the time course of DNA/RNA hybridization and RNA detection using fluorescence polarization analysis.

Using fluorescence polarization analysis, the time courses of hybridization between probe oligo-DNAs and target RNAs were measured. The RNAs were amplified using the DNA templates of Shiga toxin genes by NASBA (Nucleic Acid Sequence Based Amplification). Two DNA probes were designed for detecting the genes and they rapidly and specifically hybridized with their target RNA sequences. NASBA could be sufficiently used for the combination and DNA/RNA hybridization could be detected in the fluorescence polarization.     

Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization

Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.     

The transient-state kinetics of L-glutamate dehydrogenase. pH-dependence of the burst rate parameters.

The pH dependence of the initial transient velocity of NADPH production during the burst phase of the oxidative deamination of L-glutamate by L-glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) and NADP+ has been measured by stopped-flow spectrophotometry. These studies provide evidence that the entire pH dependence below pH 8.26 arises from reaction steps contributing to V of the burst with an apparent pKa of 8.1 +/- 0.1. The data are consistent with a model in which the formation of the first enzyme-coenzyme-substrate ternary complex on the reaction path equilibrates rapidly and in which the pH-dependent steps are mechanistically close to and may include the catalytic hydrogen transfer itself. At pH 8.87, there is evidence that L-glutamate binds less tightly to the enzyme and to the enzyme-NADP+ complex than at lower pH values.     

Physiochemical studies on interactions between DNA and RNA polymerase. Ultraviolet absorption measurements.

The interaction between Escherichia coli RNA polymerase and a restriction fragment of coliphage T7 DNA containing four promoter sites for the coli enzyme has been studied by difference uv absorption spectroscopy in a low ionic strength buffer containing 10 mm MgCl2 and 50 mM KCl. The binding of the enzyme to the DNA is accompanied by a hyperchromic shift which shows a maximum around 260 nm, and increases with increasing temperature in the temperature range studied (4-40 degrees C). Measurements were also carried out with whole T7 DNA and a restriction fragment containing no promoter site. A comparison of the results obtained with the various DNAs suggests that the binding of an RNA polymerase to a promoter site in the low ionic strength medium causes the disruption of a short segment of the DNA helix, of the order of ten pairs; the binding of an enzyme molecule to a promotor site appears to have a cooperative effect on the binding of the enzyme molecules to adjacent non-promoter sites with concomitant disruption of DNA base pairs.     

DNA in situ hybridization on polytene chromosomes of Simulium sanctipauli at loci relevant to insecticide resistance

A DNA technique for in situ hybridization developed by Kumar & Collins (1994) for use on polytene chromosomes of adult Anopheles mosquitoes (Diptera: Culicidae) was modified for use with Simulium larval salivary gland chromosomes (Diptera: Simuliidae). Cloned fragments of several Simulium genes (coding for aspartate amino transferase, cytochrome P450 and DNA polymerase) were successfully mapped physically by assigning specific band locations in Simulim sanctipauli V. & D. This represents the first attempt at locating genes beyond the resolution of linkage to inversions in any blackfly species.     

In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded 35S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.