DNA Interactive:The DNA Structure

DNA STRUCTURE

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DNA Is Composed of Polynucleotide Chains:


Schematic model of the double helix

The most importnt featur of DNA is that it is usually composed of two polynucleotde chains twistd around each other in the form of a double helix. The upper part presents the structure of the double helix shown in a schematc form. Note that if inverted 180°, the double helix looks superficially the same, due to the complementary nature of the two DNA strands. The space-filling model of the double helix, in the lower part, shows the components of the DNA molecule and their relative positions in the helical structure. The backbone of each strand of the helix is composed of alternating sugar and phosphate residues; the bases project inward but are accessible through the major and minor grooves.

Whole Genome Shotgun Sequencing (WGS)

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In contrast to the hierarchical BAC by BAC approach, which relies on the availability of genetic and physical maps for success, WGS is based on the strategy of sequencing a vast number of random genomic clones followed by intensive computer-based analysis of the DNA sequences which identifies matching sequences in different clones. This permits the assembly of a chromosomal DNA sequence, in principle without other map resources. As with the HS approach, overlapping clones are required, but since the clones are destined for direct sequence analysis, only vectors that contain small to medium inserts are normally used. Hence once the overlaps have been identified, the entire sequence is assembled. WGS was the approach adopted by the privately funded human genome initiative.
Although WGS remains somewhat controversial for sequencing complex genomes of ‘higher’ organisms because of the problems associated with repeat sequences and heterozygosity, it is a widely used approach. The number of complex genomes sequenced by this WGS is increasing and includes the fruit fly Drosophila, mosquito (anopheles), mouse, puffer fish, dog and grapevine. However, in some cases, such as the silk worm genome project, the WGS method has resulted in many seemingly irresolvable gaps in the genome and so the BAC-based hierarchical ordering of clones was used to close the gaps.Advances in computational analysis of WGS sequences suggest that the problems caused by repeat sequences could be overcome, hence the approach can be expected to gain more ground in future genome projects.

Mapping and Sequencing Strategies

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The Human Genome Project aimed to produce four types of map: physical, genetic, DNA sequence and gene. Physical and genetic maps provide essential anchor points and frameworks to align DNA sequences and assign genes. A high-resolution physical map based on the analysis of overlapping DNA clones represents the actual distance in DNA base pairs between genetic markers and other landmarks. However, the ultimate physical map is the DNA sequence itself. Low-resolution physical maps are generated from techniques such as somatic cell hybridisation and fluorescence in situ hybridisation; these methods are also applicable for assigning genes to chromosomes.
There are two approaches to genome sequencing: whole genome shotgun sequencing (WGS) and the more labour-intensive hierarchical shotgun sequencing (HS). In simple organisms such as bacteria and viruses, where the chromosomes are haploid and very little repeat sequence occurs, or for sequencing individual human genes, WGS works well. In contrast, for eukaryotic genomes, where repeat sequences often abound, including the human genome (>50% repeats), and there is considerable heterozygosity, it has been argued that HS offers advantages over WGS, and this was the approach adopted by the publicly funded International Human Genome Sequencing Consortium.

Mapping and Identifying Genes

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There are two main mapping approaches to identifying genes on chromosomes: genetic mapping and physical mapping. Genetic mapping relies on observing the recombination frequencies between pairs of polymorphic markers segregating within families. A genetic map is constructed by analysing several pairs of polymorphic genetic markers. The genetic distance between the markers is determined by their recombination frequency and measured in centimorgans (cM), where 1cM equates to 1% recombination, rather than number of nucleotides. For any pair of marker loci, the smaller the recombination frequency, the shorter is the genetic distance between the markers. The process of constructing a genetic map is called linkage analysis; this aims to determine whether or not a pair of gene loci tend to be co-inherited or separated by recombination. At one time, linkage analysis in humans was a slow process because of the dearth of polymorphic markers.However, the discovery of variable numbers of tandem repeat polymorphisms (VNTRs) and the production of genome wide panels of polymorphic microsatellites in the 1990s rendered linkage analysis one of the major tools in the Human Genome Project. Additionally, a number of physical mapping techniques were developed that enabled
genes to be identified and localised purely on the basis of their physical positions along the chromosomes; this became known as ‘reverse genetics’ and later ‘positional cloning’.

Pyrosequencing

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Rapid PCR sequencing has also been made possible by the use of pyrosequencing, which can be regarded as a second-generation sequencing method without the need for cloning in E. coli or any host cell. In one format of pyrosequencing, a PCR template is hybridised to an oligonucleotide and incubated with DNA polymerase, ATP sulfurylase, luciferase and apyrase. During the reaction, the first of the four dNTPs are added and, if incorporated, release pyrophosphate (PPi), hence the name ‘pyrosequencing’. The ATP sulfurylase converts the PPi to ATP, which drives the luciferase-mediated conversion of luciferin to oxyluciferin to generate light. Apyrase degrades the resulting component dNTPs and ATP. This is followed by another round of dNTP addition. A resulting pyrogram provides an output of the sequence. The method provides short reads very quickly and is especially useful for the determination of mutations or discovery of single nucleotide polymorphisms. Another pyrosequencing format involves direct analysis of DNA fragments and this system allows the rapid sequencing of entire genomes by the ‘shotgun’ approach. First genomic DNA is randomly sheared and ligated to linker sequences that permit individual molecules captured on the surface of a bead to be amplified while isolated within an emulsion droplet. A very large collection of such beads is arrayed in the 1.6 million wells of a fibre-optic slide. The micro-array is presented sequentially with each of the four dNTPs and the amount of incorporation is monitored by luminometric detection as before. The second-generation Roche 454 Genome Sequencer FLX is reportedly able to produce 100 Mb of sequence with 99.5% accuracy for individual reads averaging over 250 bases in length. Once again, the derived sequences can be downloaded automatically to databases and manipulated using a variety of bioinformatics resources.

Automated DNA Sequencing

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Advances in fluorescent labelling chemistry have led to the development of high-throughput automated sequencing techniques. Essentially, most systems involve the use of dideoxynucleotides labelled with different fluorochromes (often referred to as dye terminators). The advantage of this modification is that since a different label is incorporated with each ddNTP, it is unnecessary to perform four separate reactions. Therefore, the four chain-terminated products are run on the same track of a denaturing electrophoresis gel. Each product with its base-specific dye is excited by a laser and the dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions which are detected by a chargecoupled device (CCD) and the sequence is interpreted by a computer. The advantages of the techniques include real-time detection of the sequence. In addition, the lengths of sequence that may be analysed are in excess of 500 bp. Capillary electrophoresis is increasingly being used for the detection of sequencing products. This is where liquid polymers in thin capillary tubes are used, obviating the need to pour sequencing gels and requiring little manual operation. This substantially reduces the electrophoresis run times and allows high throughput to be achieved. A number of large-scale sequence facilities are now fully automated, allowing the rapid acquisition of sequence data. Automated sequencing for genome projects is usually based on cycle sequencing using instruments such as the ABI PRISM 3700 DNA Analyzer. This can be formatted to produce simultaneous reads from 384-well cycle sequencing reaction plates. The derived nt sequences are downloaded automatically to databases and manipulated using a variety of bioinformatics resources.

PCR Cycle SequencingPCR Cycle Sequencing

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One of the most useful methods of sequencing PCR amplicons is termed PCR cycle sequencing. This is not strictly a PCR, since it involves linear amplification with a single primer. Approximately 20 cycles of denaturation, annealing and extension take place. Radiolabelled or fluorescently labelled dideoxynucleotides are then introduced in the final stages of the reaction to generate the chain-terminated extension products. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in a single sequencing run

Sequencing Double-stranded DNA

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It is also possible to undertake direct DNA sequencing from doublestranded molecules such as plasmid cloning vectors and PCR amplicons. The double-stranded DNA must be denatured prior to annealing with
primer. In the case of plasmids, an alkaline denaturation step is sufficient. However, for PCR amplicons this is more problematic. Unlike plasmids, amplicons are short and reanneal rapidly. Denaturants such as formamide and dimethyl sulfoxide have been used to prevent the reannealing of PCR strands following their separation. Another strategy is to bias the amplification towards one strand by using a primer ratio of 100:1, which also overcomes this problem to a certain extent.
It is possible physically to separate and retain one PCR product strand by incorporating a molecule such as biotin into one of the primers. Following PCR, the strand that contains the biotinylated primer may be removed by affinity chromatography with streptavidin-coated magnetic beads, leaving the complementary PCR strand. This magnetic affinity purification provides single-stranded DNA derived from the PCR amplicon and, although somewhat time consuming, it does provide high-quality single-stranded DNA for sequencing.

Dideoxynucleotide Chain Terminators

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The reaction mixture is then divided into four aliquots, representing the four dNTPs, A, C, G and T. Using the adenine (A) tube as an example, in addition to all of the dNTPs being present in the mix, an analogue of dATP is added [2',3'-dideoxyadenosine triphosphate (ddATP)], which is similar to A except that it has no 30-hydroxyl group. Since a 5' to 3' phosphodiester linkage cannot be formed without a 3'-hydroxyl group, the presence of the ddATP will terminate the growing chain. The situation for tube C is identical except that ddCTP is added; similarly, the G and T tubes contain ddGTP and ddTTP, respectively.
Since the incorporation of a ddNTP rather than a dNTP is a random event, the reaction will produce new molecules varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequence are generated, each terminating at a different type of base, but all having a common 5' end (the primer). The four labelled and chainterminated samples are then denatured by heating and loaded next to each other on a polyacrylamide gel for electrophoresis. Electrophoresis is performed at approximately 70 C in the presence of urea, to prevent renaturation of the DNA, since even partial renaturation alters the rates of migration of DNA fragments. Very thin, long electrophoresis gels are used for maximum resolution over a wide range of fragment lengths. After electrophoresis, the positions of radioactive DNA bands on the gel are determined by autoradiography. Since every band in the lane from the ddATP sample must contain molecules which terminate at adenine and that those in the ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesised strand from the autoradiogram. Under ideal conditions, sequences of approximately 300 DNA bases can be read from one gel.

DNA SEQUENCING

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The determination of the order or sequence of nucleotide bases along a length of DNA is one of the central techniques in molecular biology and has played the key role in genome mapping and sequencing projects.
Two basic techniques have been developed for efficient DNA sequencing, one based on an enzymatic method frequently termed Sanger sequencing, after its developer, and a chemical method, Maxam and Gilbert sequencing, named for the same reason. For large-scale DNA analysis, Sanger sequencing and its variants are by far the most effective methods and many commercial kits are available for its use. However, there are certain occasions, such as the sequencing of short oligonucleotides, where the Maxam and Gilbert method is still more appropriate.
One absolute requirement for Sanger sequencing is that the DNA to be sequenced is in a single-stranded form. Traditionally this demanded that the DNA fragment of interest be cloned into the specialised bacteriophage vector M13, which is naturally single stranded. Although M13 is still widely used, the advent of the PCR has provided a rapid means to amplify a region of any genome or cDNA for which primer sequences are available and generate the corresponding nucleotide sequence. This has led to an explosion in DNA sequence information and has provided much impetus for polymorphism discovery by resequencing regions of the genome from individuals.
The Sanger method is simple and elegant and in many ways mimics the natural ability of DNA polymerase to extend a growing nucleotide chain based on an existing template. Initially the DNA to be sequenced is allowed to hybridise with an oligonucleotide primer, which is complementary to a sequence adjacent to the 3' side of DNA within a vector such as M13 (or within an amplicon in the case of PCR). The oligonucleotide will then act as a primer for synthesis of a second strand of DNA, catalysed by DNA polymerase. Since the new strand is synthesised from its 5' end, virtually the first DNA to be made will be complementary to the DNA to be sequenced. One of the deoxyribonucleoside triphosphates (dNTPs) which must be provided for DNA synthesis is radioactively labelled with P or S and so the newly synthesised strand will be radiolabelled.

Reverse Transcriptase PCR (RT-PCR)

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RT-PCR is an extremely useful variation of the standard PCR which permits the amplification of specific mRNA transcripts from very small biological samples without the need for the rigorous extraction procedures associated with mRNA purification for conventional cloning purposes. Conveniently, the dNTPs, buffer, Taq polymerase, oligonucleotide primers, reverse transcriptase (RT) and the RNA template are added together to the reaction tube. The reaction is heated to 37 C which allows the RT to work and permits the production of a cDNA copy of the RNA strands that anneal to one of the primers in the mix. Following ‘first strand synthesis’, a normal PCR is carried out to amplify the cDNA product, resulting in ‘second strand synthesis’, and subsequently a dsDNA product is amplified as usual. The choice of primer for the first strand synthesis depends on the experiment. If amplification of all mRNAs in the cell extract is required, then an oligo dT primer that would anneal to all the polyA tails can be used. If a specific cDNA is sought, then a coding region-specific primer can be used with success, otherwise a random primer could be used. The method is fast, accurate and simple to perform. It has many applications, such as the assessment of transcript levels in different cells and tissues (when combined with Q-PCR). When combined with allele-specific primers, it also allows the amplification of cDNA from single chromosomes. RT-PCR is widely used as a diagnostic tool in microbiology and virology.

PCR Primer Design and Bioinformatics

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The specificity of the PCR lies in the design of the two oligonucleotide primers. These not only have to be complementary to sequences flanking the target DNA but also must not be self-complementary or bind each other to form dimers, since both prevent authentic DNA amplification. They also have to be matched in their GC content and have similar annealing temperatures and be incapable of amplifying unwanted genomic sequences. Manual design of primers is time consuming and often hit or miss, although equations such as the following are still used to derive the annealing temperature (Ta) for each primer:
4(G + C) + 2(A + T) = Tm
where Tm is the melting temperature of the primer/target duplex and G, C, A and T are the numbers of the respective bases in the primer. In general, Ta is set 3–5 1C lower than Tm. On occasions, secondary or primer dimer bands may be observed on the electrophoresis gel in addition to the authentic PCR product. In such situations, Touchdown or Hot start regimes may help. Alternatively, raising Ta closer to Tm can enhance the specificity of the reaction.
The increasing use of bioinformatics resources such as Oligo, Generunner and Primer Design Assistant16 in the design of primers makes the design and the selection of reaction conditions much more straightforward. These computer-based resources allow the sequences to be amplified, primer length, product size, GC content, etc., to be input and following analysis, provide a choice of matched primer sequences. Indeed, the initial selection and design of primers without the aid of bioinformatics would now be unnecessarily time consuming. Finally, before ordering or synthesising the primers, it is wise to submit proposed sequences to a nucleotide sequence search program such as BLAST, which can be used to interrogate GenBank or other comprehensive public DNA sequence databases to increase confidence that the reaction will be specific for the intended target sequence.

THE POLYMERASE CHAIN REACTION

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In some respects, the PCR can be regarded as a form of molecular cloning, since it is a technique analogous to the DNA replication process that takes place in cells and the outcome is the same, namely the generation of new DNA molecules based exactly upon the sequence of the existing ones. PCR is a laboratory technique that is currently a mainstay of molecular biology. One of the reasons for the global adoption of the PCR is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Indeed, combined with the relevant bioinformatics resources for the design of oligonucleotide primers and for determination of the required experimental conditions, it provides a rapid means for DNA identification and analysis.
One problem with early PCR reactions was that the temperature needed to denature the DNA also denatured the DNA polymerase. However, the availability of a thermostable DNA polymerase enzyme isolated from the thermophilic bacterium Thermus aquaticus, found in hot springs, provided the means to automate the reaction. Taq DNA polymerase has a temperature optimum of 72 C and survives prolonged exposure to temperatures as high as 96 C and so is still active after each of the denaturation steps.
The PCR is often used to amplify a fragment of DNA from a complex mixture of starting material usually termed the template DNA. However, in contrast to conventional cell-based cloning, PCR does require knowledge of the DNA sequences which flank the fragment of DNA to be amplified (target DNA). From this sequence information, two oligonucleotide primers are chemically synthesised, each complementary to a stretch of DNA to the 3' side of the target DNA, one oligonucleotide for each of the two DNA strands. For many applications PCR has replaced the traditional DNA cloning methods as it fulfils the same function, the production of large amounts of DNA from limited starting material; however, this is achieved in a fraction of the time needed to clone a DNA fragment. Although not without its drawbacks, the PCR is a remarkable development which has changed the approach of many scientists to the analysis of nucleic acids and continues to have a profound impact on core genomic and genetic analysis.

cDNA libraries

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cDNA libraries

Detection of recombinant clones

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Detection of recombinant clones

DNA Libraries

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‘DNA library’ is the term used to describe a collection of recombinant clones or DNA molecules generated from a specific source of DNA. There are two main types of DNA library which are very distinct in their origin and purpose. DNA from a nucleated cell, whatever the tissue source, from a specific organism is used to make a ‘genomic’ library. The idea of a general genomic library is to produce a set of clones that contain enough DNA fragments so that the entire genome of the organism is represented. Variations of genomic DNA libraries such as chromosome-specific libraries that were prepared from chromosomes sorted by flow cytometry10 were employed in the Human Genome Project in an attempt to shorten the path between the starting DNA and generation of the genome map. The second type is the cDNA library, which is made from mRNA that has been reverse transcribed by the enzyme reverse transcriptase. Reverse transcriptase produces complementary DNA (or cDNA) fragments which are then cloned into a vector. Therefore, unlike a genomic DNA library, a cDNA library is representative of the expressed genes in a particular cell or tissue type. Thus a skeletal muscle cDNA library contains sequences expressed in the muscle tissue at the time the mRNA was harvested. cDNA libraries are particularly useful for cloning sequences where there is biological information. For example, it is known that mammalian skeletal muscle produces high levels of phosphoglucomutase (PGM1) enzyme activity, hence PGM1 cDNA clones are expected (and found) to be well represented in skeletal muscle cDNA libraries.

Identifying Clones

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Bacterial clones containing the sought after recombinant vectors can be identified by hybridisation with specific radioactively labelled or enzyme-labelled cDNA or genomic DNA probes or alternatively by the immunodetection of protein products (using specialised expression vectors which allow a cloned foreign cDNA to be transcribed to express its protein product). Both approaches are technically straightforward. Both involve the transfer of bacterial colonies from a master agar plate on to carefully orientated nitrocellulose or nylon membranes. The cells are then lysed and the DNA (or protein) from the lysed colonies is immobilised on the membrane, which is used for the probing step. Recombinant colonies can be detected as spots on X-ray film either by autoradiography or enzyme-generated chemiluminescence. The spots on the X-ray film can then be aligned with the agar master plate allowing the correct colonies to be picked. For protein detection in expression vectors, antibody probes are employed and in a manner analogous to an ELISA test. The antibody probe is conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. It is the activity of the bound enzyme on its chemiluminescence or chromogenic substrate that reveals the position of the recombinant colonies.

Library Screening

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There are several methods of screening for transformed colonies that contain recombinant vectors. For example, where the cloning site in the vector lies within an antibiotic resistance gene, successful integration of the insert will lead to inactivation of the resistance gene and recombinant colonies can be identified by a technique known as replica plating. In this method, the pattern of colonies in the original Petri dish is printed on to a nutrient agar plate containing the selective antibiotic. The position of the recombinant colonies, i.e. those that fail to grow on the selective antibiotic, is noted so that they can be picked from the master plate. In other vectors, a method called blue/white selection can be performed. In blue/white selection, successful integration of a foreign DNA molecule in the vector destroys an enzyme gene (the LacZ gene of b-galactosidase) that otherwise forms a blue product when the transformed colonies are exposed to the substrate X-gal. Thus recombinant colonies are white and non-recombinants are blue.

Replica plating to detect recombinant plasmids

The Cloning Process

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The recombinant vector molecule must be introduced into its ‘matching’ host cell in order to replicate and produce multiple copies. The process by which DNA is introduced into the host cell is known as bacterial transformation. Since ‘naked’ vector DNA is hydrophilic and the bacterial cell wall is normally impermeable to such molecules, the host cell must be made ‘competent’ by treatment with calcium chloride in the early log phase of growth. This causes the cell to become permeable to chloride ions. When competent cells are mixed with DNA and heat shocked at 42 1C, the swollen cells are able to take up the naked DNA molecules. It is believed that only a single DNA molecule is permitted to enter any single cell. Thus individual colonies of transformed bacteria grow single recombinant vector molecules. The bacterial cells are usually grown on selective media so that only transformants survive to form colonies. Thus, for example, if the vector contains an ampicillin resistance gene and the cells are grown on ampicillin-containing media, only the cells containing the vector will form colonies.

Genetic Code Table

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Genetic Code

Genes and Proteins

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Cregor Mendel might have been surprised to learn that most genes contain nothing more than instructiccss for aseesnbling proteins. He might have asked what proteine could possibly have to do with the color of a flower, the shape ofa leaf, a human blood type, or the sex of a newborn baby. The answer is that proteins have everything to do with theee things. Remember that many proteins are enzymes, which catalyze and regulate chemical reactions. A gene that codes for an enzyme to produce pigment can control the color of a flower. Another gene produces an enzyme specialized Ibr the production of red blood cell surface antigen. This molecule determines your blood type. Genes for certain proteins can regulate the rate and pattern of growth throughout an organism, controlling its size and shape. In short, proteins are microscopic tools, each specifically designed to build or operate a component of a living cell.

Genes and Proteins

The Roles of RNA and DNA

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You can compare the different rolee played by DNA and RNA molecules in directing protein synthesis to the two types of plans used by builders. A master plan has all the information needed to construct a building. But builders never bring the valuable master plan to the building site, where it might be damaged or lost. Instead, they prepare inexpensive, disposable copies of the master plan called blueprints. The master plan is safely stored in an office. and the blueprints are taken to the job sita Similarly, the cell uses the vita] DNA “master plsn to prepare RNA bluepnnts. The DNA molecule remains within the safety of the nucleus, while RNA molecules go to the protein-building sites in the cytoplasm the ribosomes.

RNA Editing

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Like a writer’s first draft, many RNA molecules require a bit of editing before they are ready to go into action. Remember that an RNA molecule is produced by copying DNA. Surprisingly, the DNA of eukaryotic genes contains sequences of nucteotidea, called Introns, that are not involved in coding for proteins. The DNA sequences that code for proteins are called exons because they are ‘expresaeC in the synthesis of proteins. When RNA molecules are formed, both the introns and the emns are copied from the DNA. However, the introna are cut out of RNA molecules while they are still in the nucleus. The remaining exona are then spliced back together to form the final mRNA. Why do ceLls use energy to make a large RNA molecule and then throw parts of it away? That’s a good question, and biologists still do not have a cmplete aoawer to it Some RNA molecules may he cut and spliced in different ways in different tissues. making it poasible for a single gene to produce several different forms of RNA. Introna and exoos may aLso play a role in evolut ion. This would make it possible for very small changes in DNA aequences to have dramatic effects in geoe expression.

RNA Editing

DNA TOPOLOGY

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As DNA is a flexibl structur its exact molecular paramete are function of both surrounding ionic environment and nature of the DNAbinding protens with which it is complexd. But if the two ends are covalently linked to form a circular DNA molecule and if there are no interruptions in the sugar phosphate backbones of the two strands, then the absolute number of times the chains can twist about each other cannot change. Because their ends are free, linear DNA molecules can freely rotate to accommodate changes in the number of times the two chains of the double helix twist about each other. Such a covalently closed, circular DNA is said to be topologically constrained. Despite these constraints, DNA participates in numerous dynamic processes in the cell. For example, the two strands of the double helix, which are twisted around each other, must rapidly separate in order for DNA to be duplicated and to be transcribed into RNA. Thus, understanding the topology of DNA and how the cell both accommodates and exploits topological constraints during DNA replication, transcription, and other chromosomal transactions is of fundamental importance in molecular biology.

RNA STRUCTURE

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We now turn our attention to RNA, which differs from DNA in three respects. First, the backbone of RNA contains ribose rather than 2'-deoxyribose. That is, ribose has a hydroxyl group at the 2' position. Second, RNA contains uracil in place of thymine. Uracil has the same single-ringed structure as thymine, except that it lacks the 5' methyl group. Thymine is in effect 5'methyl-uracil. Third, RNA is usually found as a single polynucleotide chain. Except for the case of certain viruses, RNA is not the genetic material and does not need to be capable of serving as a template for its own replication. Rather, RNA functions as the intermediate, the mRNA, between the gene and the protein-synthesizing machinery. Another function of RNA is as an adaptor, the tRNA, between the codons in the mRNA and amino acids. RNA can also play a structural role as in the case of the RNA components of the ribosome. Yet another role for RNA is as a regulatory molecule, which through sequence complementarity binds to, and interferes with the translation of, certain mRNAs. Finally, some RNAs are enzymes that catalyze essential reactions in the cell. In all of these cases, the RNA is copied as a single strand off only one of the two strands of the DNA template, and its complementary strand does not exist. RNA is capable of forming long double helices, but these are unusual in nature.

RNA Structure