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65-440: X-ray crystallography is not common for nucleic acids alone, since neither DNA nor RNA readily form crystals. This is due to the greater degree of intrinsic disorder and dynamism in nucleic acid structures and the negatively charged (deoxy)ribose-phosphate backbones, which repel each other in close proximity. Therefore, crystallized nucleic acids tend to be complexed with a protein of interest to provide structural order and neutralize

130-416: A purine or pyrimidine nucleobase (sometimes termed nitrogenous base or simply base ), a pentose sugar , and a phosphate group which makes the molecule acidic. The substructure consisting of a nucleobase plus sugar is termed a nucleoside . Nucleic acid types differ in the structure of the sugar in their nucleotides–DNA contains 2'- deoxyribose while RNA contains ribose (where the only difference

195-514: A DNA copy, the DNA generated is truncated at the positions of reaction because the enzyme is blocked by the adducts. The collection of DNA molecules of various truncated lengths therefore informs the frequency of reaction at every base position, which reflects the structure profile along the RNA. This is traditionally assayed by running the DNA on a gel , and the intensity of bands inform the frequency of observing

260-441: A barrierless Friedel-Crafts reaction. LASER probing targets both single-stranded and double-stranded residues as long as they are solvent accessible. Because hydroxyl radical probing requires synchrotron radiation to measure solvent accessibility of RNA in vivo , it is hard to apply hydroxyl radical probing to footprint RNA in cells for many laboratories. In contrast, LASER probing utilizes a hand-held UV lamp (20 W) for excitation, it

325-436: A cylation analyzed by p rimer e xtension, or SHAPE , takes advantage of reagents that preferentially modify the backbone of RNA in structurally flexible regions. Reagents such as N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7) react with the 2'-hydroxyl group to form adducts on the 2'-hydroxyl of the RNA backbone. Compared to the chemicals used in other RNA probing techniques, these reagents have

390-437: A high-throughput fashion. The carbodiimide moiety can also form covalent adducts at exposed nucleobases, which are uracil , and to a smaller extent guanine , upon nucleophilic attack by a deprotonated N. They react primarily with N3 of uracil and N1 of guanine modifying two sites responsible for hydrogen bonding on the bases. 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho- p -toluene sulfonate, also known as CMCT or CMC,

455-429: A ligand, as well as compare that response to analogous ligands. This assay is commonly used in dynamic studies, specifically when examining riboswitches . Nucleotide analog interference mapping (NAIM) is the process of using nucleotide analogs, molecules that are similar in some ways to nucleotides but lack function, to determine the importance of a functional group at each location of an RNA molecule. The process of NAIM

520-526: A regular double helix, and can adopt highly complex three-dimensional structures that are based on short stretches of intramolecular base-paired sequences including both Watson-Crick and noncanonical base pairs, and a wide range of complex tertiary interactions. Nucleic acid molecules are usually unbranched and may occur as linear and circular molecules. For example, bacterial chromosomes, plasmids , mitochondrial DNA , and chloroplast DNA are usually circular double-stranded DNA molecules, while chromosomes of

585-623: A sample requires taking many images (often referred to as electron micrographs) and averaging over those images in a process called single-particle reconstruction . This is a computationally intensive process. Cryo-EM is a newer, less perturbative version of transmission electron microscopy (TEM). It is less perturbative because the sample is not dried onto a surface, this drying process is often done in negative-stain TEM , and because Cryo-EM does not require contrast agent like heavy metal salts (e.g. uranyl acetate or phoshotungstic acid) which also may affect

650-499: A sequencing experiment Site directed incorporation results indicate positions of importance where when running on a gel, functional RNAs that have the analog incorporated at that position will have a band present, but if the analog results in non-functionality, when the functional RNA molecules are run on a gel there will be no band corresponding to that position on the gel. This process can be used to evaluate an entire area, where analogs are placed in site specific locations, differing by

715-416: A single RNA molecule, which enables one to obtain more information per read (for a read of 150 nt, typically two to three mutation sites, rather than zero to one truncation sites), determine structures of low-abundance RNAs, and identify subpopulations of RNAs with alternative secondary structures. DMS-MaPseq uses a thermostable group II intron reverse transcriptase (TGIRT) that creates a mutation (rather than

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780-471: A single nucleotide, then when functional RNAs are isolated and run on a gel, all areas where bands are produced indicate non-essential nucleotides, but areas where bands are absent from the functional RNA indicate that inserting a nucleotide analog in that position caused the RNA molecule to become non-functional Nucleic acid Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides , which are

845-953: A structure related to 1,2-dicarbonyls, and was the first in this category used extensively for the chemical probing of RNA. Kethoxal causes the modification of guanine, specifically altering the N1 and the exocyclic amino group (N2) simultaneously by covalent interaction. Glyoxal , methylglyoxal, and phenylglyoxal, which all carry the key 1,2-dicarbonyl moiety, all react with free guanines similar to kethoxal, and can be used to probe unpaired guanine bases in structured RNA. Due to their chemical properties, these reagents can permeate readily into cells and can therefore be used to assay RNAs in their native cellular environments. Light-Activated Structural Examination of RNA (LASER) probing utilizes UV light to activate nicotinoyl azide (NAz), generating highly reactive nitrenium cation in water, which reacts with solvent accessible guanosine and adenosine of RNA at C-8 position through

910-403: A truncation at each position. Recent approaches use high-throughput sequencing to achieve the same purpose with greater throughput and sensitivity. The reactivity profile can be used to study the degree of structure at particular positions for specific hypotheses, or used in conjunction with computational algorithms to produce a complete experimentally supported structure model. Depending on

975-414: A truncation) in the cDNA when it encounters a base methylated by DMS, but otherwise it reverse transcribes with high fidelity. Sequencing the resulting cDNA identifies which bases were mutated during reverse transcription; these bases cannot have been base-paired in the original RNA. DMS modification can also be used for DNA, for example in footprinting DNA-protein interactions. S elective 2′- h ydroxyl

1040-422: Is a chemical that can be used to modify nucleic acids in order to determine secondary structure. Reaction with DMS adds a methyl adduct at the site, known as methylation . In particular, DMS methylates N1 of adenine (A) and N3 of cytosine (C) , both located at the site of natural hydrogen bonds upon base-pairing. Therefore, modification can only occur at A and C nucleobases that are single-stranded, base paired at

1105-528: Is a single molecule that contains 247 million base pairs ). In most cases, naturally occurring DNA molecules are double-stranded and RNA molecules are single-stranded. There are numerous exceptions, however—some viruses have genomes made of double-stranded RNA and other viruses have single-stranded DNA genomes, and, in some circumstances, nucleic acid structures with three or four strands can form. Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components:

1170-456: Is also used for the chemical probing of RNA structure. EDC is able to permeate into cells and is thus used for direct in-cell probing of RNA in their native environments. Some 1,2-di carbonyl compounds are able to react with single-stranded guanine (G) at N1 and N2, forming a five-membered ring adduct at the Watson-Crick face. 1,1-Dihydroxy-3-ethoxy-2- butanone , also known as kethoxal , has

1235-406: Is considered a distinct molecular species . Examples include the addition of sodium bisulfite to an aldehyde to give a sulfonate . It can be considered as a single product resulting from the direct combination of different molecules which comprises all atoms of the reactant molecules. Adducts often form between Lewis acids and Lewis bases . A good example is the formation of adducts between

1300-420: Is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecules. Adduct In chemistry , an adduct (from Latin adductus  'drawn toward'; alternatively, a contraction of "addition product") is a product of a direct addition of two or more distinct molecules , resulting in a single reaction product containing all atoms of all components. The resultant

1365-404: Is kept extremely cold by a liquid nitrogen bath. Upon this freezing process, water molecules in the sample do not have enough time to form hexagonal lattices as found in ice, and therefore the sample is preserved in a glassy water-like state (also referred to as a vitrified ice ), making these samples easier to image using the electron beam. An advantage of cryo-EM over x-ray crystallography is that

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1430-551: Is much easier to apply LASER probing for in vivo studying RNA solvent accessibility. This chemical probing method is light-controllable, and probes solvent accessibility of nucleobase, which has been shown to footprint RNA binding proteins inside cells. In-line probing does not involve treatment with any type of chemical or reagent to modify RNA structures. This type of probing assay uses the structure dependent cleavage of RNA; single stranded regions are more flexible and unstable and will degrade over time. The process of in-line probing

1495-465: Is often used to determine changes in structure due to ligand binding. Binding of a ligand can result in different cleavage patterns. The process of in-line probing involves incubation of structural or functional RNAs over a long period of time. This period can be several days, but varies in each experiment. The incubated products are then run on a gel to visualize the bands. This experiment is often done using two different conditions: 1) with ligand and 2) in

1560-411: Is one of four types of molecules called nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes genetic information. This information specifies the sequence of the amino acids within proteins according to the genetic code . The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription. Within cells, DNA

1625-453: Is organized into long sequences called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and archaea) store their DNA only in

1690-561: Is the nucleotide , each of which contains a pentose sugar ( ribose or deoxyribose ), a phosphate group, and a nucleobase . Nucleic acids are also generated within the laboratory, through the use of enzymes (DNA and RNA polymerases) and by solid-phase chemical synthesis . Nucleic acids are generally very large molecules. Indeed, DNA molecules are probably the largest individual molecules known. Well-studied biological nucleic acid molecules range in size from 21 nucleotides ( small interfering RNA ) to large chromosomes ( human chromosome 1

1755-404: Is the adducts of ethylene or carbon monoxide of CuAlCl 4 . The latter is a solid with an extended lattice structure . Upon formation of the adduct, a new extended phase is formed in which the gas molecules are incorporated (inserted) as ligands of the copper atoms within the structure. This reaction can also be considered a reaction between a base and a Lewis acid with the copper atom in

1820-460: Is the most commonly used carbodiimide for RNA structure probing. Similar to DMS, it can be detected by reverse transcription followed by gel electrophoresis or high-throughput sequencing. As it is reactive towards G and U, it can be used to complement the data from DMS probing experiments, which inform A and C. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide , also known as EDC, is a water-soluble carbodiimide that exhibits similar reactivity as CMC, and

1885-431: Is the presence of a hydroxyl group ). Also, the nucleobases found in the two nucleic acid types are different: adenine , cytosine , and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA. The sugars and phosphates in nucleic acids are connected to each other in an alternating chain (sugar-phosphate backbone) through phosphodiester linkages. In conventional nomenclature ,

1950-412: Is to insert a single nucleotide analog into a unique site. This can be done by transcribing a short RNA using T7 RNA polymerase , then synthesizing a short oligonucleotide containing the analog in a specific position, then ligating them together on the DNA template using a ligase. The nucleotide analogs are tagged with a phosphorothioate, the active members of the RNA population are then distinguished from

2015-478: The University of Tübingen , Germany. He discovered a new substance, which he called nuclein and which - depending on how his results are interpreted in detail - can be seen in modern terms either as a nucleid acid- histone complex or as the actual nucleid acid. Phoeber Aaron Theodor Levene, an American biochemist determined the basic structure of nucleic acids. In the early 1880s, Albrecht Kossel further purified

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2080-459: The methyl groups on the tin and the ethyl groups on oxygen. But when the Lewis base is tetrahydrofuran, steric repulsion is reduced. The ECW model can provide a measure of these steric effects. Compounds or mixtures that cannot form an adduct because of steric hindrance are called frustrated Lewis pairs . Adducts are not necessarily molecular in nature. A good example from solid-state chemistry

2145-660: The monomer components: a 5-carbon sugar , a phosphate group and a nitrogenous base . The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose , the polymer is RNA; if the sugar is deoxyribose , a variant of ribose, the polymer is DNA. Nucleic acids are chemical compounds that are found in nature. They carry information in cells and make up genetic material. These acids are very common in all living things, where they create, encode, and store information in every living cell of every life-form on Earth. In turn, they send and express that information inside and outside

2210-574: The nucleus , and for the presence of phosphate groups (related to phosphoric acid). Although first discovered within the nucleus of eukaryotic cells, nucleic acids are now known to be found in all life forms including within bacteria , archaea , mitochondria , chloroplasts , and viruses (There is debate as to whether viruses are living or non-living ). All living cells contain both DNA and RNA (except some cells such as mature red blood cells), while viruses contain either DNA or RNA, but usually not both. The basic component of biological nucleic acids

2275-599: The sequence of nucleotides . Nucleotide sequences are of great importance in biology since they carry the ultimate instructions that encode all biological molecules, molecular assemblies, subcellular and cellular structures, organs, and organisms, and directly enable cognition, memory, and behavior. Enormous efforts have gone into the development of experimental methods to determine the nucleotide sequence of biological DNA and RNA molecules, and today hundreds of millions of nucleotides are sequenced daily at genome centers and smaller laboratories worldwide. In addition to maintaining

2340-563: The GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. Deoxyribonucleic acid (DNA) is a nucleic acid containing the genetic instructions used in the development and functioning of all known living organisms. The chemical DNA

2405-640: The Lewis acid borane and the oxygen atom in the Lewis bases, tetrahydrofuran (THF): BH 3 ·O(CH 2 ) 4 or diethyl ether : BH 3 ·O(CH 3 CH 2 ) 2 . Many Lewis acids and Lewis bases reacting in the gas phase or in non-aqueous solvents to form adducts have been examined in the ECW model . Trimethylborane , trimethyltin chloride and bis(hexafluoroacetylacetonato)copper(II) are examples of Lewis acids that form adducts which exhibit steric effects . For example: trimethyltin chloride, when reacting with diethyl ether, exhibits steric repulsion between

2470-504: The RNA can be protected from the reagents not only by local structure but also by a binding protein over that position. This has led some work to use chemical probing to also assay protein-binding. As hydroxyl radicals are short-lived in solution, they need to be generated upon experiment. This can be done using H 2 O 2 , ascorbic acid, and Fe(II)-EDTA complex. These reagents form a system that generates hydroxyl radicals through Fenton chemistry . The hydroxyl radicals can then react with

2535-407: The absence of ligand. Cleavage results in shorter band lengths and is indicative of areas that are not basepaired, as basepaired regions tend to be less sensitive to spontaneous cleavage. In-line probing is a functional assay that can be used to determine structural changes in RNA in response to ligand binding. It can directly show the change in flexibility and binding of regions of RNA in response to

2600-502: The advantage of being largely unbiased to base identity, while remaining very sensitive to conformational dynamics. Nucleotides which are constrained (usually by base-pairing) show less adduct formation than nucleotides which are unpaired. Adduct formation is quantified for each nucleotide in a given RNA by extension of a complementary DNA primer with reverse transcriptase and comparison of the resulting fragments with those from an unmodified control. SHAPE therefore reports on RNA structure at

2665-665: The atoms usually observed in NMR, and because nucleic acid double helices are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are H or proton NMR , C NMR , N NMR , and P NMR . Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space. Parameters taken from

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2730-433: The binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which resonances are shifted upon binding of the other molecule. Cryogenic electron microscopy (cryo-EM) is a technique that uses an electron beam to image samples that have been cryogenically preserved in an aqueous solution. Liquid samples are pipetted on small metallic grids and plunged into a liquid ethane/propane solution which

2795-520: The carbons to which the phosphate groups attach are the 3'-end and the 5'-end carbons of the sugar. This gives nucleic acids directionality , and the ends of nucleic acid molecules are referred to as 5'-end and 3'-end. The nucleobases are joined to the sugars via an N -glycosidic linkage involving a nucleobase ring nitrogen ( N -1 for pyrimidines and N -9 for purines) and the 1' carbon of the pentose sugar ring. Non-standard nucleosides are also found in both RNA and DNA and usually arise from modification of

2860-446: The cell nucleus. From the inner workings of the cell to the young of a living thing, they contain and provide information via the nucleic acid sequence . This gives the RNA and DNA their unmistakable 'ladder-step' order of nucleotides within their molecules. Both play a crucial role in directing protein synthesis . Strings of nucleotides are bonded to form spiraling backbones and assembled into chains of bases or base-pairs selected from

2925-456: The chemical reagent used, some reagents, e.g. hydroxyl radicals, would cleave the RNA molecule instead. The result in the truncated DNA is the same. Some reagents, e.g. DMS, sometimes do not block the reverse transcriptase, but trigger a mistake at the site in the DNA copy instead. These can be detected when using high-throughput sequencing methods, and is sometimes employed for improved results of probing as mutational profiling (MaP). Positions on

2990-621: The cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. Ribonucleic acid (RNA) functions in converting genetic information from genes into the amino acid sequences of proteins. The three universal types of RNA include transfer RNA (tRNA), messenger RNA (mRNA), and ribosomal RNA (rRNA). Messenger RNA acts to carry genetic sequence information between DNA and ribosomes, directing protein synthesis and carries instructions from DNA in

3055-424: The double helix does not have a compact interior and does not fold back upon itself. NMR is also useful for investigating nonstandard geometries such as bent helices , non-Watson–Crick basepairing, and coaxial stacking . It has been especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt complex conformations such as stem-loops and pseudoknots . NMR is also useful for probing

3120-465: The double-helix structure of DNA . Experimental studies of nucleic acids constitute a major part of modern biological and medical research , and form a foundation for genome and forensic science , and the biotechnology and pharmaceutical industries . The term nucleic acid is the overall name for DNA and RNA, members of a family of biopolymers , and is a type of polynucleotide . Nucleic acids were named for their initial discovery within

3185-590: The end of a helix, or in a base pair at or next to a GU wobble pair , the latter two being positions where the base-pairing can occasionally open up. Moreover, since modified sites cannot be base-paired, modification sites can be detected by RT-PCR, where the reverse transcriptase falls off at methylated bases and produces different truncated cDNAs. These truncated cDNAs can be identified through gel electrophoresis or high-throughput sequencing. Improving upon truncation-based methods, DMS mutational profiling with sequencing (DMS-MaPseq) can detect multiple DMS modifications in

3250-425: The eukaryotic nucleus are usually linear double-stranded DNA molecules. Most RNA molecules are linear, single-stranded molecules, but both circular and branched molecules can result from RNA splicing reactions. The total amount of pyrimidines in a double-stranded DNA molecule is equal to the total amount of purines. The diameter of the helix is about 20 Å . One DNA or RNA molecule differs from another primarily in

3315-632: The five primary, or canonical, nucleobases . RNA usually forms a chain of single bases, whereas DNA forms a chain of base pairs. The bases found in RNA and DNA are: adenine , cytosine , guanine , thymine , and uracil . Thymine occurs only in DNA and uracil only in RNA. Using amino acids and protein synthesis , the specific sequence in DNA of these nucleobase-pairs helps to keep and send coded instructions as genes . In RNA, base-pair sequencing helps to make new proteins that determine most chemical processes of all life forms. Nucleic acid was, partially, first discovered by Friedrich Miescher in 1869 at

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3380-450: The inactive members, the inactive members then have the phosphorothioate tag removed and the analog sites are identified using gel electrophoresis and autoradiography. This indicates a functionally important nucleotide, as cleavage of the phosphorothioate by iodine results in an RNA that is cleaved at the site of the nucleotide analog insert. By running these truncated RNA molecules on a gel, the nucleotide of interest can be identified against

3445-422: The individual nucleotide level. This data can be used as input to generate highly accurate secondary structure models. SHAPE has been used to analyze diverse RNA structures, including that of an entire HIV-1 genome. The best approach is to use a combination of chemical probing reagents and experimental data. In SHAPE-Seq SHAPE is extended by bar-code based multiplexing combined with RNA-Seq and can be performed in

3510-428: The negative charge. Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA . As of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy. Nucleic acid NMR uses similar techniques as protein NMR, but has several differences. Nucleic acids have a smaller percentage of hydrogen atoms, which are

3575-409: The nucleic acid molecules. Hydroxyl radicals attack the ribose/deoxyribose ring and this results in breaking of the sugar-phosphate backbone. Sites under protection from binding proteins or RNA tertiary structure would be cleaved by hydroxyl radical at a lower rate. These positions would therefore show up as absence of bands on the gel, or low signal through sequencing. Dimethyl sulfate , known as DMS,

3640-515: The nucleid acid substance and discovered its highly acidic properties. He later also identified the nucleobases . In 1889 Richard Altmann created the term nucleic acid – at that time DNA and RNA were not differentiated. In 1938 Astbury and Bell published the first X-ray diffraction pattern of DNA. In 1944 the Avery–MacLeod–McCarty experiment showed that DNA is the carrier of genetic information and in 1953 Watson and Crick proposed

3705-481: The nucleobase level (2-3 angstroms) up to tertiary structure motifs (greater than a nanometer). RNA chemical probing uses chemicals that react with RNAs. Importantly, their reactivity depends on local RNA structure e.g. base-pairing or accessibility. Differences in reactivity can therefore serve as a footprint of structure along the sequence. Different reagents react at different positions on the RNA structure, and have different spectra of reactivity. Recent advances allow

3770-515: The nucleus to ribosome . Ribosomal RNA reads the DNA sequence, and catalyzes peptide bond formation. Transfer RNA serves as the carrier molecule for amino acids to be used in protein synthesis, and is responsible for decoding the mRNA. In addition, many other classes of RNA are now known. Artificial nucleic acid analogues have been designed and synthesized. They include peptide nucleic acid , morpholino - and locked nucleic acid , glycol nucleic acid , and threose nucleic acid . Each of these

3835-431: The samples are preserved in their aqueous solution state and not perturbed by forming a crystal of the sample. One disadvantage, is that it is difficult to resolve nucleic acid or protein structures that are smaller than ~75 kilodaltons , partly due to the difficulty of having enough contrast to locate particles in this vitrified aqueous solution. Another disadvantage is that to attain atomic-level structure information about

3900-412: The simultaneous study of the structure of many RNAs (transcriptome-wide probing) and the direct assay of RNA molecules in their cellular environment (in-cell probing). Structured RNA is first reacted with the probing reagents for a given incubation time. These reagents would form a covalent adduct on the RNA at the site of reaction. When the RNA is reverse transcribed using a reverse transcriptase into

3965-478: The spectrum, mainly NOESY cross-peaks and coupling constants , can be used to determine local structural features such as glycosidic bond angles, dihedral angles (using the Karplus equation ), and sugar pucker conformations. For large-scale structure, these local parameters must be supplemented with other structural assumptions or models, because errors add up as the double helix is traversed, and unlike with proteins,

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4030-483: The standard nucleosides within the DNA molecule or the primary (initial) RNA transcript. Transfer RNA (tRNA) molecules contain a particularly large number of modified nucleosides. Double-stranded nucleic acids are made up of complementary sequences, in which extensive Watson-Crick base pairing results in a highly repeated and quite uniform nucleic acid double-helical three-dimensional structure. In contrast, single-stranded RNA and DNA molecules are not constrained to

4095-477: The structure of the biomolecule. Transmission electron microscopy, as a technique, utilizes the fact that samples interact with a beam of electrons and only parts of the sample that do not interact with the electron beam are allowed to 'transmit' onto the electron detection system. TEM, in general, has been a useful technique in determining nucleic acid structure since the 1960s. While double-stranded DNA (dsDNA) structure may not traditionally be considered structure, in

4160-472: The typical sense of alternating segments of single- and double-stranded regions, in reality, dsDNA is not simply a perfectly ordered double helix at every location of its length due to thermal fluctuations in the DNA and alternative structures that can form like g-quadruplexes . CryoEM of nucleic acid has been done on ribosomes, viral RNA, and single-stranded RNA structures within viruses. These studies have resolved structural features at different resolutions from

4225-672: Was discovered in 1869, but its role in genetic inheritance was not demonstrated until 1943. The DNA segments that carry this genetic information are called genes. Other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Along with RNA and proteins, DNA is one of the three major macromolecules that are essential for all known forms of life. DNA consists of two long polymers of monomer units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands are oriented in opposite directions to each other and are, therefore, antiparallel . Attached to each sugar

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