Molecular hybridization of ribonucleic acid with a large excess of deoxyribonucleic acid

When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA-RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA-RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2xSSC and 3xSSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2xSSC buffer. Rate-constants for DNA-RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA-RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90-95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.     

An assay for quantitative nucleic acid hybridization on membrane filters

A quantitative nucleic acid hybridization assay based on a 6-mm-diameter nitrocellulose membrane filter and only 20 microliters of hybridization mixture per determination is described. As a consequence of the small ratio of hybridization volume to membrane surface, the hybridization rates reached in this system are higher than those obtained at volume to surface ratios of conventional protocols, allowing even RNA at very low concentrations to complete hybridization. This advantage, together with the high reproducibility and hybrid stability obtained with the assay, increases the ability of the filter hybridization technique to analyze quantitatively minute amounts of RNA.     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA.

Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans.     

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.     

Xylo-Configured oligonucleotides (XNA,xylo nucleic acid): synthesis of conformationally restricted derivatives and hybridization towards DNA and RNA complements

Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 2'-deoxy-2'-fluoro-beta-D-xylofuranosyl nucleotide monomer, a novel conformationally locked 2'-amino-2'-deoxy-2'-N,4'-C-methylene-beta-D-xylofuranosyl nucleotide monomer, and a known 2'-deoxy-beta-D-xylofuranosyl nucleotide monomer (XNA monomers) have been synthesized and their hybridization towards DNA and RNA complements studied. Thermal denaturation studies of nine-mer mixed-base sequences composed of a mixture of XNA monomers and DNA monomers revealed preferential hybridization towards RNA complements relative to DNA complements. For 14-mer homo-thymine XNAs containing thirteen XNA monomers, stable complexes towards single-stranded DNA and RNA were formed at pH 7. Gel-shift experiments revealed these complexes to involve at least two XNA strands per DNA or RNA target strand.     

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage phi X174. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples.     

昆虫小器官RNA荧光原位杂交技术

【目的】在昆虫基因表达和功能研究中,RNA原位杂交技术越来越受到青睐。该技术不仅能定性定量反应基因表达的时空特异性,而且能在细胞水平上检测基因表达的调控模式。为了将该技术更好地在昆虫小器官研究中运用,我们以果蝇幼虫翅芽为例优化了改技术。【方法】解剖果蝇3龄幼虫翅芽进行原位杂交实验。【结果】我们发现影响原位杂交结果的因素十分复杂,包括取材时期,探针的合成,预杂交/杂交的时间和温度,清洗时间,适当的对照等。通过RNA荧光原位杂交实验,我们揭示了调控细胞记忆的trithorax基因在3龄翅芽广泛表达,并且受到转录因子Optomotor-blind的负调控。【结论】这一技术方法为研究昆虫小器官的基因表达和调控提供了便捷手段。     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

The Impact of Viral RNA on Assembly Pathway Selection

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T = 3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T = 3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.     

Estimation of gene reiteration from hybridization kinetics in moderate deoxyribonucleic acid excess.

Theoretical calculations were carried out to clarify how the DNA/RNA or the DNA/cDNA (complementary DNA) ratio in the hybridization reaction mixture affects the kinetics of DNA-RNA or DNA-cDNA reassociation, and theoretical formulae were derived as a function of these ratios. From these formulae, it was found that the DNA/RNA of the DNA/cDNA ratio did not much affect the initial reaction rates of hybridization, but greatly affected the terminal value for the extent of hybrid formation. Therefore the results obtained when one normalizes the experimental data for hydridization and derives the reiteration frequency from a number called the 'half Cot' (Cot 1/2) are not accurate, especially in the presence of a moderate excess of DNA. A simple method for the estimation of gene reiteration was demonstrated that did not use the half Cot value in the determination. This simple method is useful even if DNA-RNA or DNA/cDNA hybridization are done with a moderate excess of DNA. With mouse myeloma cells as a model system, the gene reiteration of the 28S rRNA cristron was determined.     

Ribonucleic acid synthesis termination protein rho function: effects of conditions that destabilize ribonucleic acid secondary structure

The dependence fo rate of adenosine 5'-triphosphate (ATP) hydrolysis catalyzed by ribonucleic acid (RNA) synthesis termination protein rho from Escherichia coli with T7 RNA as cofactor is used to probe the nature of the interaction between rho and RNA. In general, reaction conditions that destabilize the secondary structure of the RNA enhance its cofactor activity. This is indicated by the effects of MgCl2 concentration, spermidine, temperature, dimethyl sulfoxide, and pretreatment of the RNA with formaldehyde. These results suggest that a functional interaction between rho and RNA depends either on the presence of a sufficiently large single-stranded region in the RNA or on the ability of rho to unwind double helices in the RNA. It is also shown that changes in reaction conditions that increase RNA secondary structure and decrease the rho protein adenosine triphosphate phosphohydrolase (rhoATPase) activity with isolated T7 RNA also decrease the stringency of rho action in RNA synthesis termination. On the other hand, monovalent salts decrease rhoATPase activity with isolated T7 RNA and binding of rho to T7 RNA independently of the MgCl2 concentration and thus the relative stability of the RNA secondary structure.     

Measurement of proviral genes in uninfected and avian myeloblastosis virus-infected cells by hybridization with 3H-labeled complementary DNA probe excess.

Viral RNA (vRNA) from avian myeloblastosis virus or DNA from virus-infected and uninfected cells was hybridized with [3H]DNA complementary to viral RNA ([3H]cDNA) under conditions of [3H]cDNA excess. When [3H]cDNA was used to drive the hybridization reaction with vRNA, a rate constant of 33.2 liters/mol-s was obtained. The same rate constant was obtained when vRNA excess was used as the driver. The specific activities of the [3H]DNA probe, estimated from kinetic measurements of the hybridization reaction and from the amount of [3H]cDNA in hybrid form at equilibrium, were 9.1 and 8.6 cpm/pg, respectively. DNA isolated from uninfected cells contained five or six copies of proviral DNA per cell genome. DNA isolated from erythrocytes infected with avian myeloblastosis virus had an additional five or six viral genes added to the cell genome, and the virus-infected target cell (myeloblasts) contained about 15 additional copies of proviral DNA per cell. The use of excess [3H]cDNA probe is an easy and accurate method to quantify the frequency of proviral DNA sequences in cell DNA and to measure a small amount (40 to 200 pg) of vRNA. Probe excess hybridization offers a number of advantages over other procedures and these are discussed.     

Quantitation of endogenous liver apolipoprotein B mRNA editing

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.