Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

Studies towards the development of chemically synthesized non-radioactive biotinylated nucleic acid hybridization probes.

Non-radioactive nucleic acid hybridization probes have been constructed in which the reporter group is long chain biotin chemically linked to a basic macromolecule (histone H1, cytochrome C or polyethyleneimine). The modified basic macromolecule which carries many biotin residues can, in turn, be covalently linked to nucleic acids (DNA) via the bifunctional cross-linking reagents, glutaraldehyde, 1,2,7,8-diepoxyoctane, bis (succinimidyl) suberate or bis (sulfonosuccinimidyl) suberate. This provides a very sensitive probe by which as little as between 10-50fg of target DNA can be visualized using dot-blot hybridization procedures in conjunction with avidin or streptavidin enzyme conjugates.     

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).     

Thermodynamic limits on the size and size distribution of nucleic acids synthesized in vitro: the role of pyrophosphate hydrolysis.

The free-energy change of phosphodiester bond formation from nucleoside triphosphates is more favorable than with nucleoside diphosphates as substrates. Base-stacking interactions can make significant contributions to both delta G degrees ' values. Pyrophosphate hydrolysis when it accompanies the former reaction dominates all thermodynamic considerations. Three experimental situations are discussed in which high-molecular-weight polynucleotides are synthesized without a strong driving force for covalent bond formation. For one of these, a kinetic scheme is presented which encompasses an early narrow Poisson distribution of chain lengths with ultimate passage to a disperse equilibrium population of chain sizes. Hydrolytic removal of pyrophosphate expands the time scale for this undesirable process by a factor of 10(9), while it enormously elevates the thermodynamic ceiling for the average degrees of polymerization in the other two examples. The electron micrographically revealed broad size population from an early study of partial replication of a T7 DNA template is found to adhere (fortuitously) to a disperse most probable representation. Some possible origins are examined for the branched structures in this product, as well as in a later investigation of replication of this nucleic acid. The achievement of both very high molecular weights and sharply peaked size distributions in polynucleotides synthesized in vitro will require coupling to inorganic pyrophosphatase action as in vivo.     

Architectural editing: determining the fate of newly synthesized membrane proteins

Many integral membrane proteins exist on the plasma membrane as part of multicomponent complexes. In addition to correctly transporting newly synthesized proteins from their site of synthesis in the endoplasmic reticulum to the plasma membrane, the cell must possess mechanisms to ensure that the complexes expressed on the cell surface are accurately assembled. The cell appears to accomplish this feat by superimposing a set of constraints on the newly synthesized membrane proteins whereby the structure and state of assembly of the protein determine its intracellular fate. These processes impose a dramatic level of post-translational regulation on the expression of surface membrane protein complexes. By and large, the cell uses these mechanisms to dispose of, or "edit out," newly synthesized proteins that are not correctly assembled or folded. This review will describe current views of the processes of architectural editing, with an emphasis on the regulation of cell surface expression of the multicomponent T-cell antigen receptor complex.     

Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions

We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.     

Mfold web server for nucleic acid folding and hybridization prediction

The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as 'MFOLDROOT'.     

A microcomputer program for analysis of nucleic acid hybridization data

The study of nucleic acid hybridization is facilitated by computer mediated fitting of theoretical models to experimental data. This paper describes a non-linear curve fitting program, using the `Patternsearch' algorithm, written in BASIC for the Apple II microcomputer. The advantages and disadvantages of using a microcomputer for local data processing are discussed.     

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.     

Use of bacterial luciferase for determining monoamine oxidase activity

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.     

A comprehensive model for the diffusion and hybridization processes of nucleic acid probes in fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of microorganisms. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes (MPs) move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different MPs to diffuse across multiple cellular barriers, as well as the reaction time associated with the formation of the duplex molecular probe-RNA, were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 7.94 × 10² – 4.39 × 10³ s), whereas for animal cells, the limiting step should be the diffusion of the probe through the bulk (1.8–5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis for a more rational development of FISH protocols in the future.