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Competition in DNA hybridization to RNA

Competition in DNA hybridization to RNA


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After we separate the two DNA strands, what makes one of the two strands combine with the labelled mRNA strand instead of the old complementary strand?


In this type of experiment the concentration of the mRNA will be greater than the concentration of the starting DNA.

When the DNA strands separate, the DNA and mRNA will be in competition for the complementary strand. Using really high concentrations of mRNA compared to the DNA will give it advantage to recombine with a higher frequency and give a higher ratio of the DNA-mRNA combination.

It is all a question of ratio.


Nucleic acid hybridization

In molecular biology, hybridization (or hybridisation) is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. [1] Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory (generally by raising the surrounding temperature) will cause the molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.

DNA replication and transcription of DNA into RNA both rely upon nucleotide hybridization, as do molecular biology techniques including Southern blots and Northern blots, [2] the polymerase chain reaction (PCR), and most approaches to DNA sequencing.


PATTERNS OF RNA SYNTHESIS IN T5-INFECTED CELLS I. AS STUDIED BY THE TECHNIQUE OF DNA-RNA HYBRIDIZATION-COMPETITION *

The RNA-labeling patterns obtained after T5 infection of Escherichia coli F agree with the patterns of protein labeling published by McCorquodale and Buchanan. 1 Three distinct classes of RNA formed sequentially during the period of viral development can be recognized by the DNA-RNA hybridization-competition technique. Class I RNA is formed within 5 minutes after the beginning of viral metabolism and corresponds to the RNA synthesized in response to infection with the 8 per cent segment of T5 DNA. Protein synthesis directed by this 8 per cent segment is required in some capacity for the cessation of class I synthesis and the beginning of the synthesis of class II at 4 to 5 min after infection. Class III RNA synthesis begins between 9 and 12 minutes. Its appearance is prevented when chloramphenicol is added immediately after complete expression of class I functions.


DNA probes: applications of the principles of nucleic acid hybridization

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.


Ribonucleic acid synthesis during microcyst formation in Myxococcus xanthus: characterization by deoxyribonucleic acid-ribonucleic acid hybridization

The technique of deoxyribonucleic acid-ribonucleic acid (RNA) hybridization was used to compare the RNA synthesized during vegetative growth and microcyst formation in Myxococcus xanthus. All classes of RNA, including ribosomal RNA, were synthesized during microcyst formation. The results indicate that the ribosomal RNA synthesized during microcyst formation was indistinguishable from that made during vegetative growth. Hybridization competition experiments demonstrated that certain messenger RNA species are synthesized only during vegetative growth, whereas others are synthesized only during microcyst formation. The synthesis of a new species of RNA polymerase does not appear to be responsible for differential transcription during morphogenesis in M. xanthus since the rifampicin sensitivity of transcription was conserved during microcyst formation.


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Methods

Examine evolution of self-replicating DNA and RNA molecules under conditions likely present in primordial earth.

Setting (initial conditions)

Our proposed evolutionary dynamics occur in tide pools, likely common in primordial earth. Tide pools provide weakly dispersive, spatially constrained liquid environment containing multiple monomer species generated by prebiotic chemistry [40, 42,43,44]. We assume the nucleotides were synthesized within the tidal pool via reactions initially at equilibrium. Thus, as products of these reactions were consumed in formation of polynucleotides, more would be synthesized. Thus, to the extent that the reaction pathways “competed” for substrate, those that generated nucleotide most favourable for polymer formation would become the primary consumers.

We then subject the pool to regular, diurnal cycles near the melting temperatures of polynucleotides. The average Archean ocean temperature is estimated to be 26-35 °C but may have reached 70 °C in some locations [41, 45] and likely varied over space and time. Other initial conditions such as the reducing atmosphere, the pH, salinity, and ion concentrations in the primordial oceans [27] may have contributed to the dynamics of prebiotic chemistry and polymer autocatalysis.

Within these conditions, the diurnal thermal cycle applied regular and predictable perturbations to the environment. The critical role of cyclical thermodynamic fluctuations (as opposed to stochastic fluctuations or a thermodynamically constant state) in developing information and converting energy to order (decreased entropy) has been widely recognized [46].

We hypothesize higher daytime temperatures, similar to Polymerase Chain Reactions (PCR), provided sufficient thermal energy to melt hydrogen bonds causing the separation of polymer strands. Simultaneously, daytime UV-irradiation from sunlight could promote nucleobase formation [4]. As thermal energy decreased during night hours, each single strand could replicate as the free monomers formed hydrogen bonds with counterparts on the template strand, leading to covalent bonds between adjacent monomers and the synthesis of a daughter strand [14].

Evolution by natural selection requires heritable phenotypic variation and environmental constraints that limit proliferation such that the replicative success of an organism is governed by its properties and those of competing organisms. We propose these conditions are met as self-replicating RNA or its ancestral macromolecules, within the physical constraint of a tide pool, compete for available monomers. In addition, we note the diurnal cycle also imposes constraints on replicative speed. That is, successful completion of each daughter strand synthesis must occur prior to onset of daytime temperatures and strand separation. Thus, these prebiotic evolutionary dynamics would impose selection for fidelity and speed of replication, efficiency of substrate utilization, and stability (persistence) under local environmental conditions.

Finally, fluctuating water levels in tidal pools permitted influx of new monomers and efflux of replicators. This latter resulted in dispersal of replicators so that successful variants could colonize new pools that lack self-replicating species or were populated by replicators with inferior properties. This weak coupling of subpopulations could accelerate and promote natural selection similar to Wright’s shifting balance theory [47].

Mathematical models and computer simulations

The mathematical models and details of computer simulations are discussed in detail in references [26]. All code used in the simulations is available from the link provided below.


Competition in DNA hybridization to RNA - Biology

a State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China
E-mail: [email protected]

b Wallace H. Coulter Department of Biomedical Engineering, Emory University School of Medicine, Emory University, Atlanta, Georgia 30322, USA
E-mail: [email protected]

Abstract

RNA imaging in living animals helps decipher biology and creates new theranostics for disease treatment. Due to their low delivery efficiency and high background, however, fluorescence probes for in situ RNA imaging in living mice have not been reported. We develop a new cell-targeting fluorescent probe that enables RNA imaging in living mice via an in vivo hybridization chain reaction (HCR). The minimalistic Y-shaped design of the tripartite DNA probe improves its performance in live animal studies and serves as a modular scaffold for three DNA motifs for cell-targeting and the HCR circuit. The tripartite DNA probe allows facile synthesis with a high yield and demonstrates ultrasensitive RNA detection in vitro. The probe also exhibits selective and efficient internalization into folate (FA) receptor-overexpressed cells via a caveolar-mediated endocytosis mechanism and produces fluorescence signals dynamically correlated with intracellular target expressions. Furthermore, the probe exhibits specific delivery into tumor cells and allows high-contrast imaging of miR-21 in living mice. The tripartite DNA design may open the door for intracellular RNA imaging in living animals using DNA-minimal structures and its design strategy can help future development of DNA-based multi-functional molecular probes.


Advantages of Hybridization

(1) Two species combine to form the best of the organism.

(2) Hybrid plants are physically uniform. This is manly advantageous for farmers who harvest with machines, but it is usually not a big deal for small greenhouse gardeners.

(3) Hybridization help in getting different species of animals.

(4) They pass along favorable traits and prolong the survival of threatened or endangered species.

(5) They (hybrids) often show greater vigor and faster growth

(6) Hybrids can have up to 25 (twenty-five) percent higher yields.

(7) They result in the formation of organisms that possess various qualities such as disease resistance, stress resistance, and so on.

(8) In animals such as humans, hybridization help in the detection of the presence of amplified genes in cancer and to map out their location.

(9) Hybridization creates room for a better adaptation of new species.


Analysis of RNA by Northern and Slot Blot Hybridization

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting) unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions these methods are outlined in a Support Protocol.


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