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8.1: Nucleic Acids - Structure and Function

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    Search Fundamentals of Biochemistry

    Introduction to Nucleic Acids

    Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life. The nucleic acids consists of two major macromolecules, Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that carry the genetic instructions for the development, functioning, growth and reproduction of all known organisms and viruses. Both consist of polymers of a sugar-phosphate-sugar backbone with organic heterocyclic bases attached to the sugars. The sugar in DNA is deoxyribose while in RNA it is ribose. DNA contain four bases, cytosine and thymine (pyrimidine bases) and guanine and adenine (purine bases). DNA in vivo consist of two antiparallel strands intertwined to form the iconic DNA double-stranded helix. RNA is single stranded but may adopt many secondary and tertiary conformations not unlike that of a protein. Figure \(\PageIndex{1}\) below shows a low resolution comparison of the structure of DNA and RNA.

    Difference_DNA_RNA-EN.svg
    Figure \(\PageIndex{1}\): Low resolution comparison of the structure of DNA and RNA. https://commons.wikimedia.org/wiki/F...DNA_RNA-EN.svg. Creative Commons Attribution-Share Alike 3.0 Unported license.

    The biological function of DNA is quite simple, to carry and protect the genetic code. Its structure serves that purpose well. In the next section, we will study the functions of RNA, which are much more numerous and complicated. The structure of RNA has evolved to serve those added functions.

    The core structure of a nucleic acid monomer is the nucleoside, which consists of a sugar residue + a nitrogenous base that is attached to the sugar residue at the 1′ position as shown in Figure \(\PageIndex{2}\) below. The sugar utilized for RNA monomers is ribose, whereas DNA monomers utilize deoxyribose that has lost the hydroxyl functional group at the 2′ position of ribose. For the DNA molecule, there are four nitrogenous bases that are incorporated into the standard DNA structure. These include the Purines: Adenine (A) and Guanine (G), and the Pyrimidines: Cytosine (C) and Thymine (T). RNA uses the same nitrogenous bases as DNA, except for Thymine. Thymine is replaced with Uracil (U) in the RNA structure.

    When one or more phosphate groups are attached to a nucleoside at the 5′ position of the sugar residue, it is called a nucleotide. Nucleotides come in three flavors depending how many phosphates are included: the incorporation of one phosphate forms a nucleoside monophosphate, the incorporation of two phosphates forms a nucleoside diphosphate, and the incorporation of three phosphates forms a nucleoside triphosphate as shown in Figure \(\PageIndex{2}\) below.

    bases_sidesandtides.svg
    Figure \(\PageIndex{2}\):The Monomer Building Blocks of Nucleic Acids. The site of the nitrogenous base attachment to the sugar residue (glycosidic bond) is shown in red.

    DNA and RNA Hydrogen-bonded structures

    Figure \(\PageIndex{3}\) below shows a "flattened" structure of double-stranded B-DNA that best shows the backbone and hydrogen bonded base pairs between two antiparallel strands of the DNA. Unlike the protein α-helix, where the R-groups of the amino acids are positioned to the outside of the helix, in the DNA double-stranded helix, the nitrogenous bases are positioned inward and face each other. The backbone of the DNA is made up of repeating sugar-phosphate-sugar-phosphate residues. Bases fit in the double helical model if pyrimidine on one strand is always paired with purine on the other. From Chargaff’s rules, the two strands will pair A with T and G with C. This pairs a keto base with an amino base, a purine with a pyrimidine. Two H‑bonds can form between A and T, and three can form between G and C. This third H-bond in the G:C base pair is between the additional exocyclic amino group on G and the C2 keto group on C. The pyrimidine C2 keto group is not involved in hydrogen bonding in the A:T base pair.

    Furthermore, the orientation of the sugar molecule within the strand determines the directionality of the strands. The phosphate group that makes up part of the nucleotide monomer is always attached to the 5′ position of the deoxyribose sugar residue. The free end that can accept a new incoming nucleotide is the 3′ hydroxyl position of the deoxyribose sugar. Thus, DNA is directional and is always synthesized in the 5′ to 3′ direction. Interestingly, the two strands of the DNA double helix lie in opposite directions or have a head to tail orientation.

    DNA_chemical_structure.svg
    Figure \(\PageIndex{3}\): "Flattened" Structure of DNA Madeleine Price Ball. https://en.Wikipedia.org/wiki/File:D..._structure.svg. Wikimedia Commons

    By analogy to proteins, DNA and RNA can be loosely thought to have primary and secondary structures. For a single strand, the primary sequence is just the base sequence read from the 5' to 3' end of the strand, with the bases thought of as "side chains" as illustrated in Figure \(\PageIndex{4}\) below for an RNA strand which contains U instead of T.

    DNAprimaryseqV2.png

    Figure \(\PageIndex{4}\): "Primary" sequence of a single RNA strand. https://en.Wikipedia.org/wiki/Nucleic_acid_sequence. Creative Commons Attribution-ShareAlike License

    Since it is found partnered with another molecule (strand) of DNA, the double stranded DNA, which consists of two molecules held together by hydrogen bonds, might be considered to have secondary structure (analogous to alpha and beta structure in proteins). Of course the hydrogen bonds are not between backbone atoms but between side chain bases in double stranded DNA.

    Figure \(\PageIndex{5}\) below shows an interactive iCn3D model of the the iconic structure of a short oligomer of double-stranded DNA (1BNA).

    1BNA_BaseStacking.png

    Figure \(\PageIndex{5}\): Iconic structure of a short oligomer of double-stranded DNA (1BNA). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...b5HUbmuQrCobg8

    The backbones of the antiparallel strands are magenta (chain A) and cyan (chain B). The 5' sugar-phosphate end of the of each chain are shown in spacefill and colored magenta (chain A) and cyan (chain B). The hydrogen-bonded interstrand base pairs are shown alternatively in spacefill and sticks to illustrate how the bases stack on top of each other.

    Figure \(\PageIndex{6}\) below shows types of "secondary (flat representations) and their 3D or tertiary representations found in nucleic acids.

    PrimarySeqStructureDNA.png

     

    Figure \(\PageIndex{6}\): https://en.Wikipedia.org/wiki/Nucleic_acid_sequence. Creative Commons Attribution-ShareAlike License

     

    Figure \(\PageIndex{7}\) below shows an interactive iCn3D model of the tertiary structure of the T4 hairpin loop on a Z-DNA stem (1D16).

    T4 hairpin loop on a Z-DNA stem (1D16).png

    Figure \(\PageIndex{7}\): T4 hairpin loop on a Z-DNA stem (1D16). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...8C7qBqgh8ZTJH9

    The hairpin shown is from a synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G which adopts an alternative Z-DNA conformation (which we will explore below) with a loop at one end. The thymine bases 7, 8 and 9 are generally perpendicular to one another and stack together, along with the ribose of T7.

    Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of pseudoknot in RNA (437D).

    Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot (437D).png

    Figure \(\PageIndex{8}\): Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot (437D). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ZtdeJqQXvjCKfA

    The pseudoknot has two stems that form a "helix" and two loops. The knot consists of a hairpin in the nucleic acid structure with the loop between the helices paired to another part of the nucleic acid. Pseudoknots can be found in mRNA and in ribosomal RNA and affect the translation of the RNA (decoding to instruct the synthesis of a protein sequence). RNA virus have pseudoknots which likewise affects protein synthesis as well as RNA replication. Pseudoknots also occur in DNA.

     

    Synthesis and structure of DNA

    The nucleotide that is required as the monomer for the synthesis of both DNA and RNA is nucleoside triphosphate. During the incorporation of the nucleotide into the polymeric structure, two phosphate groups, (Pi-Pi , called pyrophosphate) from each triphosphate are cleaved from the incoming nucleotide and further hydrolyzed during the reaction, leaving a nucleoside monophosphate that is incorporated into the growing RNA or DNA chain as shown in Figure \(\PageIndex{9}\) below. Incorporation of the incoming nucleoside triphosphate is mediated by the nucleophilic attack of the 3′-OH of the growing DNA polymer. Thus, DNA synthesis is directional, only occuring at the 3′-end of the molecule.

    The further hydrolysis of the pyrophosphate (Pi-Pi) releases a large amount of energy ensuring that the overall reaction has a negative ΔG. Hydrolysis of Pi-Pi ↔ 2Pi has a ΔG = -7 kcal/mol (-29 kJ/mol) and is essential to provide the overall negative ΔG (-6.5 kcal/mol, 27 kJ/mol) of the DNA synthesis reaction. Hydrolysis of the pyrophosphate also ensures that the reverse reaction, pyrophosphorolysis, will not take place removing the newly incorporated nucleotide from the growing DNA chain.

    This reaction is mediated in DNA by a family of enzymes known as DNA polymerases. Similarly, RNA polymerases are required for RNA synthesis. A more detailed description of polymerase reaction mechanisms will be covered in Chapters X and Y, covering DNA Replication and Repair, and DNA Transcription.

    nucleotide-incorporation-1024x659.png
    Figure \(\PageIndex{9}\): Nucleic Acid Synthesis: In nucleic acid synthesis, the 3’ OH of a growing chain of nucleotides attacks the α-phosphate on the next NTP to be incorporated (blue), resulting in a phosphodiester linkage and the release of pyrophosphate (PPi). The DNA polymerase further mediates the hydrolysis of the pyrophosphate preventing the reverse reaction from occurring and releasing enough energy to drive the reaction forward. The synthesis of DNA is shown in this diagram. Image modified from Michal Sobkowski

    DNA was first isolated by Friedrich Miescher in 1869. The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953,(X,Y,Z coordinates in 1954) based upon the crucial X-ray diffraction image of DNA from Rosalind Franklin in 1952, followed by her more clarified DNA image with Raymond Gosling, Maurice Wilkins, Alexander Stokes, and Herbert Wilson, and base-pairing chemical and biochemical information by Erwin Chargaff. The prior model was triple-stranded DNA.

    The realization that the structure of DNA is that of a double-helix elucidated the mechanism of base pairing by which genetic information is stored and copied in living organisms and is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one third of the 1962 Nobel Prize in Physiology or Medicine for their contributions to the discovery. (Franklin, whose breakthrough X-ray diffraction data was used to formulate the DNA structure, died in 1958, and thus was ineligible to be nominated for a Nobel Prize.)

    Watson and Crick proposed two strands of DNA – each in a right-hand helix – wound around the same axis. The two strands held together by H-bonding between the complimentary base pairs (A pairs with T and G pairs with C) as shown in Figure \(\PageIndex{10}\) below. Note that when looking from the top view, down on a DNA base pair, that the position where the base pairs attach to the DNA backbone is not equidistant, but that attachment favors one side over the other. This creates unequal gaps or spaces in the DNA known as the major groove for the larger gap, and the minor groove for the smaller gap (Figure 4.5). Based on the DNA sequence within the region, the hydrogen-bond potential created by the nitrogen and oxygen atoms present in the nitrogenous base pairs cause unique recognition features within the major and minor grooves, allowing for specific protein recognition sites to be created.

    major-minor-grooves-1024x1004.png
    Figure \(\PageIndex{10}\): The Major and Minor Grooves of DNA. Top view of an (A) A-T base pair and a (B) G-C base pair showing the formation of the major and minor groove sides of the DNA. (C) Side view of the DNA double helix with the major and minor grooves indicated. The DNA backbone is shown in green, potential nitrogen hydrogen-bonding locations are indicated in blue, and oxygen hydrogen-bonding locations in red. Figure C modified from: dullhunk

    Figure \(\PageIndex{1}\) below shows a schematic representation of available hydrogen bond donors and acceptors in the major and minor groove for TA and CG base pairs.

    Hbondmajmingrove.svg
    Figure \(\PageIndex{11}\): Available hydrogen bond donors and acceptors in the major and minor groove for TA and CG base pairs

    Figure \(\PageIndex{12}\) below shows an interactive iCn3D model of DNA showing the major and minor grooves.

    MajorMinorGroves_dsDNA.png

     

    Figure \(\PageIndex{12}\): Major and minor groves of ds-DNA. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?WQWkYi1FrM4DSY2i7

    The two sugar-phosphate backbones are shown in green and yellow. Some of the red (oxygen) and blue (nitrogen) atoms in the major grove (and to a much less extent in the minor groove) are not involved in inter-strand G-C and A-T base pairing and so would available to hydrogen bond donors with specific binding proteins that would display complementary shape and hydrogen bonds acceptors and donors.

    In addition to the major and minor grooves providing variation within the double helix structure, the axis alignment of the helix along with other influencing factors such as the degree of solvation, can give rise to three forms of the double helix, the A-form (A-DNA), the B-form (B-DNA), and the Z-form (Z-DNA) as shown in Figure \(\PageIndex{13}\) below.

    A-DNA,_B-DNA_and_Z-DNA.png
    Figure \(\PageIndex{13}\): Structures of A-DNA (left), B-DNA (middle), and Z-DNA (right). https://en.Wikipedia.org/wiki/File:A..._and_Z-DNA.png. Creative Commons Attribution-Share Alike 3.0 Unported

    Both the A- and B-forms of the double helix are right-handed spirals, with the B-form being the predominant form found in vivo. The A-form helix arises when conditions of dehydration below 75% of normal occur and have mainly been observed in vitro during X-ray crystallography experiments when the DNA helix has become desiccated. However, the A-form of the double helix can occur in vivo when RNA adopts a double stranded conformation, or when RNA-DNA complexes form. The 2′-OH group of the ribose sugar backbone in the RNA molecule prevents the RNA-DNA hybrid from adopting the B-conformation due to steric hindrance.

    The third type of double helix formed is a left-handed helical structure known as the Z-form, or Z-DNA. Within this structural motif, the phosphates within the backbone appear to zigzag, providing the name Z-DNA. In vitro, the Z-form of DNA is adopted in short sequences that alternate pyrimidine and purines and when high salinity is present. However, the Z-form has been identified in vivo, within short regions of the DNA, showing that DNA is quite flexible and can adopt a variety of conformations. A comparison of features between A-, B- and Z-form DNA is shown in Table 4.1.

    Table 4.1 Comparisons of B-form, A-form and Z‑DNA
      B-Form A-Form Z-Form
    helix sense Right Handed Right Handed Left Handed
    base pairs per turn 10 11 12
    vertical rise per bp 3.4 Å 2.56 Å 19 Å
    rotation per bp +36° +33° -30°
    helical diameter 19 Å 19 Å 19 Å

    The double stranded helix of DNA is not always stable. This is because the stair step links between the strands are noncovalent, reversible interactions. Depending on the DNA sequence, denaturation (melting) can be local or widespread and enables various crucial cellular processes to take place, including DNA replication, transcription, and repair.

    Both sequence specificity and interaction (whether covalent or not) with a small compound or a protein can induce tilt, roll and twist effects that rotate the base pairs in the x, y, or z axis, respectively as seen in Figure \(\PageIndex{14}\) , and can therefore change the helix’s overall organization. Furthermore, slide or flip effects can also modify the geometrical orientation of the helix. Hence the flip effects, and (to a lesser extent) the other above-defined movements modulate the double-strand stability within the helix or at its ends. Indeed, under physiological conditions, local DNA ‘breathing’ has been evidenced at both ends of the DNA helix and B- to Z-DNA structural transitions have been observed in internal DNA regions. These types of locally open DNA structures are good substrates for specific proteins which can also induce the opening of a ‘closed’ helix. The processes of DNA replication and repair will be discussed in more detail in Chapter 28.

    DNADestabAgents-Fig1.svg

    Figure \(\PageIndex{14}\): Localized Structural Modification of the DNA Double Helix. (a) Base pair orientation with x, y, and z axes result in different kinds of rotation (tilt, roll or twist) or slipping of the bases (slide, flip) regarding to the helix central axis. (b) Matove B-DNA with nearly 11 base pairs within one helical turn. (c) Mono- or bis-intercalation of a small molecule (shown in blue) between adjacent base pairs resulting in an unwinding of the DNA helix (orange arrow on the top) and a lengthening of the DNA helix (ΔLength) depending on the X and y Å values that are specific for a defined DNA intercalating compound. (d) Representation of the DNA bending, base flipping, or double strand opening induced by some DNA destabilizing alkylating agents (adducts shown in blue). Adapted from Calladine and Drew’s schematic box representation. Lenglet and David-Cordonnier (2010) Journal of Nucleic Acids, http://dx.doi.org/10.4061/2010/290935. Creative Commons Attribution License,

    Figure \(\PageIndex{15}\) below shows interactive iCn3D models of A-DNA (top) , B-DNA (center) and Z-DNA (bottom). (Copyright; author via source). Click the image for a popup or use the external links in column 1.

    A-DNA (440D) A-DNA_440D.png
    B-DNA (1BNA) BDNA_1BNA.png
    Z-DNA (4OCB) ZDNA-4OCB.png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{15}\): A, B and Z-DNA. Click the image for a popup or use the links in column 1

     

    We studied the structure of proteins in depth, discussing resonance in the peptide backbone, allowed backbone angles φ, ψ and ω, side chain rotamers, Ramachandran plots and different structural motifs. We also explored them dynamically using molecular dynamic simulations. We also discussed the thermodynamics of protein stability, and how stability could be altered by changing environmental factors such as solution composition and temperature.

    In contrast, our understanding of the structural parameters and the dynamics of nucleic acids is less advanced. This may seem paradoxical, especially given the apparently simplicity of the iconic structure of DNA presented in textbooks. Yet look at the types of secondary structures of nucleic acid presented and then the complicated tertiary and quaternary structures of RNA.

    The backbone of nucleic acid has a 5-membered sugar ring, which adds rigidity to the backbone, linked to another sugar ring by CH2O(PO3)O- connectors, which add some additional conformational freedom. We'll explore the effects of the pentose ring geometry in RNA and DNA in chapter section 8.3. To illustrate a yet unexplored complexity of nucleic acid structure, consider just the orientation of rings in double stranded DNA and in regions of RNA where double-stranded structures form. The variants in orientation of the hydrogen bonded base pairs and the corresponding parameters that define them are shown in Figure \(\PageIndex{16}\) below.

    basepairparameters.jpeg

    Figure \(\PageIndex{16}\): Base pair orientation and corresponding parameters in nucleic acids. http://x3dna.org/highlights/schemati...air-parameters (with permission). 2008 3DNA Nature Protocols paper (NP08), the initial 3DNA Nucleic Acids Research paper .

    Consider just two of these, the propellor and twist angles. If you examine the iCn3D models of nucleic acids presented above, you will see the the base pairs are not perfectly flat but are twisted. Larger propeller angles are associated with increased rigidity. The propellor angles for A, B and Z DNA are +18o, + 16 +/-7 o, and about 0o, respectively. The twist angles A, B and Z DNA are +33o, +36 o, and -30o, respectively. The lower the twist angle, the higher the number of base pairs per turn. This of course affects the pitch of the helix (the length of one complete turn). All of these terms should be minimized to computatationally determine the lowest energy state for a given double-stranded nucleic acid.

    Alternative Base Pairing in DNA and RNA

    A first glance at a DNA or RNA structure reveals a myriad of possible hydrogen bond donors and acceptors in the bases of the nucleic acid. Hence it should come as no surprise that a variety of alternative or noncanonical (not in the canon or dogma) intermolecular hydrogen bonds can form between and among bases, leading to alternatives to the classical Watson-Crick base pairing. In fact there are 28 possible base pairs that form two hydrogen bond between them. As structure determines function and activity, these alternative structures also influence DNA/RNA function. We will consider four different types of noncanonical base pairing: reverse Watson Crick, wobble, Hoogsteen and reverse Hoogsteen base pairs.

    In DNA, these types of noncanonical base pairs can occur when bases become mismatched in double-stranded regions. In RNA, which we will explore more fully in the Chapter 8.2, double stranded molecules form by separate RNA molecules aren't common. Instead the molecule folds on itself in 3D space to form complex tertiary structure containing regions of helical secondary structure. RNAs also form quaternary structure when bound to other nucleic acids and protein. Larger RNAs have loops with complex secondary and tertiary structures which often require noncanonical base pairing, which stabilize the alternative structures. The noncanonical structures are also important for RNA-protein interactions in the RNA region which binds proteins. If one considers RNA and protein binding as a coupled equilibrium, it should be clear that protein binding to RNA might also induce conformation changes, specifically noncanonical base pairs, in the RNA. For example, the HIV Rev peptide binds to a target site in the envelop gene of HIV (which has an RNA genome) and leads to the formation of an RNA loop with hydrogen bonding between two purines.

    Figure \(\PageIndex{17}\) below shows an interactive iCn3D model of the REV Response element RNA complexed with REV peptide (1ETF).

    REV Response element RNA complexed with REV peptide (1ETF).png

    Figure \(\PageIndex{17}\): REV Response element RNA complexed with REV peptide (1ETF). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...T8CJ3pCe986Vx9

    The peptide is shown in cyan and its arginine side chains are shown as cyan lines. There are an extraordinary number of arginines which form ion-ion interactions with the negatively charged phosphates in the major grove of this double-stranded A-RNA. The noncanonical base pairs are shown in CPK colored sticks. A wobble base, U43-G77, see below, is shown as well as three homopurine base pairs, G47-A73, G55-A58 and G48-G71. The solitary A68 base is shown projecting away from the RNA.

    Figure \(\PageIndex{18}\) below shows the Watson Crick and first set of alternative non-canonical base pairs.

    WatsonCreek_Reverse_Wobble.svg

    Figure \(\PageIndex{18}\): Some noncanonical base nucleic acid base pairs

     

    Let's look at them in more detail.

    Reverse Watson Crick: The reverse Watson-Crick AT (AU) and GC pairs can sometimes be found at the end of DNA strands and also in RNA. In forming the reverse bases pairs, the pyrimidine can rotate 180o along the axis shown and the rotate in the plane to align the hydrogen bond donors and acceptors as shown in the top part of the figure. The glycosidic bond between the N in the base and the sugar (the circled R group) are now in an "antiparallel" arrangement in the reverse base pair.

    Wobble Base Pairs

    The bases in nucleic acids can undergo tautomerization to produce forms that can base pair noncanonically. The are termed wobble base pairs and include G-T(U) base pairs from keto–enol tautomerism and A-C base pairs from amino–imino tautomerism, as illustrated in Figure 18 above.

    Figure \(\PageIndex{19}\) below shows an interactive iCn3D model of the GT Wobble Base-Pairing in Z-DNA form of d(CGCGTG) (1VTT). Two such GT pairs are found in the structure.

    GT Wobble Base-Pairing in Z-DNA form of d(CGCGTG) (1VTT).png

    Figure \(\PageIndex{19}\): GT Wobble Base-Pairing in Z-DNA form of d(CGCGTG) (1VTT). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...LtwfzyeqDCaPEA

    Water around the wobble base pairs can form hydrogen bonds and stabilize the pair if an hydrogen bond is missing.

    Figure \(\PageIndex{20}\) below shows an interactive iCn3D model of dsRNA with G-U wobble base pairs (6L0Y).

    dsRNA with G-U wobble base pairs (6L0Y).png

    Figure \(\PageIndex{20}\): dsRNA with G-U wobble base pairs (6L0Y). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...reyeD6JQM1djq6

    The structure contains many GU wobble base pairs as well as two CU base pairs between two pyrimidine bases.

    Inosine, a variant of the base adenine, can be found in RNA. It is forme by the deamination of adenosine by the enzyme adenosine deaminase. A nucleotide having inosine is named hypoxanthine. Hypoxanthine can from the wobble base pairs I-U, I-A and I-C when incorporated into RNA, as illustrate in Figure \(\PageIndex{21}\) below..

     

    WobbleswithI.svg

    Figure \(\PageIndex{21}\): Wobble bases pairs using hypoxanthine with the base inosine

    Wobble base pair interactions are especially important in the process of translation when a protein sequence is made from a messenger RNA template (which will discuss in Unit III). For that decoding process to occur, two RNA molecules, messenger RNA (mRNA) and a transfer RNA (t-RNA) covalently attached to a specific amino acid like glutamic acid, must bind to each other through a 3 base pair interaction. The 3 bases on the mRNA are called the codon, and the 3 complementary bases on the tRNA are called the anticodon. The triplet base pair are antiparallel to each other. The interaction between mRNA and tRNA are illustrate in Figure \(\PageIndex{22}\) below.

    Elongator and codon biasTrnaRrnaFig1.svg

    Figure \(\PageIndex{22}\): The wobble uridine (U34) of tRNA molecules that recognize both AAand AG-ending codons for Lys, Gln, and Glu, is modified by the addition of both a thiol (s2) and a methoxy-carbonyl-methyl (mcm5). This double modification enhances the translational efficiency of AA-ending codons. Goffena, J et al. Nat Commun 9, 889 (2018). https://doi.org/10.1038/s41467-018-03221-z. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

    The third 3' base on the mRNA is less restricted and can form noncanonical, specifically, wobble base pairs, with the 5' base in the anti-codon triplet of tRNA. The term wobble arise from the subtile conformational changes use to optimize the pairing of the triplets. Wobble bases occur much more in tRNA than other nucleic acids.

    Hoogsteen base pairing

    Flexibility in DNA allows rotation around the C1'-N glycosidic bond connecting the deoxyribose and base in DNA, allowing different orientations of AT and GC base pairs with each other. The normal "anti" orientation allows "Watson-Crick" (WC) base pairing between AT and GC base pairs while the altered rotation allows "Hoogsteen" base pairs. The different orientations for an AT base pair are shown in Figure \(\PageIndex{23}\) below.

    HoogsteenvsWCrickBasePair2018Fig1.svg

    Figure \(\PageIndex{23}\): Xu, Y., McSally, J., Andricioaei, I. et al. Modulation of Figure \(\PageIndex{xx}\)Hoogsteen dynamics on DNA recognition. Nat Commun 9, 1473 (2018). https://doi.org/10.1038/s41467-018-03516-1Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

    Hoogsteen base pairing is usually see when DNA is distorted through interactions with bound proteins and drugs that intercalate between base pairs. Figure \(\PageIndex{24}\) below shows an interactive iCn3D model of a Hoogsteen base pair embedded in undistorted B-DNA - MATAlpha2 homeodomain bound to DNA (1K61).

    A Hoogsteen base pair embedded in undistorted B-DNA - (1K61).png

    Figure \(\PageIndex{24}\): A Hoogsteen base pair embedded in undistorted B-DNA - MATAlpha2 Homeodomain bound to DNA (1K61). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SLLRv1m8HQXKcA

    The same DNA without bound protein has no Hoogsteen base pairs. To form Hoogsteen base pairs, a rotation around the glycosidic-base bond must occur. Hoogsteen base pairs between G and C can also occur on rotation but in addition, the N3 of cytosine is protonated, as shown in Figure 14 above.

    Evidence suggest that Hoogsteen base pairing may be iimportant in DNA replication, binding, damage or repair. They can induce kinking of the DNA near the major grove.

    There are also examples of reverse Hoogsteen base pairing, as shown in Figure \(\PageIndex{25}\) below.

    Hoogsteen_Rev_Hoogsteen.svg

    Figure \(\PageIndex{25}\): The reverse Hoogsteen AT base pair

    Additional Alternative Structures: Quadruplexes and Triple Helices

    Quadruplexes

    These can be formed in DNA and RNA from G-rich sequences involving tetrads of guanine bases that are hydrogen bonded. They are a bit hard to describe in words so lets first examine one particular structure.

    In human cells, telomeres (the ends of chromosomes) contain 300-8000 repeats of a simple TTAGGG sequence. The repetitive TTAGGG sequences in telomeric DNA can form quadruplexes. Figure \(\PageIndex{26}\) below shows an interactive iCn3D model of parallel quadruplexes from human telomeric DNA (1KF1). The structure contains a single DNA strand (5'-AGGGTTAGGGTTAGGGTTAGGG-3') which contains four TTAGGG repeats.

    parallel quadruplexes from human telomeric DNA (1KF1).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{26}\): parallel quadruplexes from human telomeric DNA (1KF1). (Copyright; author via source).
    Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...y5joFHDgWJQsQ6

    Rotate the model to see 3 parallel layers of quadruplexes. In each layer, 4 noncontiguous guanine bases interact with a K+ ion. Hover over the guanine bases in one layer and you will find that one layer consists of guanines 4, 10, 16 and 22, which derive from the last G in each of the repeats in the sequence of the oligomer used (5'-AGGGTTAGGGTTAGGGTTAGGG-3'). These quadruplexes certainly serve as recognition and binding site for telomerase proteins. The guanine-rich telomere sequences which can form quadruplex may also function to stabilize chromosome ends

    Quadruplex can be forme in 1 strand of a nucleic acid (as in the above model) or from 2 or 4 separate strands. They also must have at least 2 stacked triads. As in the example above, single stranded sections can form intramolecular G-quadrplex from a GmXnGmXoGmXpGm sequence, where m is he number of Gs in each short setment (3 in the structure above). If a segment is longer than others, a G might be in a loop.

    Triple Helices

    These structures can occur in DNA (and also RNA) that contain homopurine and homopyrimidine sequences that have a mirror repeat symmetry. Hence they can occur naturally. A mirror repeat contains a center of symmetry on a single strand. Here is an example: 5'-GCATGGTACG-3'.

    They can also occur when a third single-strand DNA (called a triplex forming oligonucleotide or TFO) binds to a double-stranded DNA. The TFOs bind through Hoogsteen base pairing in the major grove of the ds-DNA. They can bind tightly and specifically and in a parallel or antiparallel fashion. Specific and locally higher concentrations of divalent cations or positively charged polyamines like spermine act to stabilized the extra negative charge density from the binding of a third polyanionic DNA strand.

    An example of a triple helix system that has been studied in vitro is shown in Figure \(\PageIndex{27}\) below.

    TripleHelices1_Fig2.svg

    Figure \(\PageIndex{27}\): Intermolecular triplex formation and their oligonucleotide sequences (where “•” and “-” indicates Hoogsteen and Watson–Crick base pairings, respectively). Inset: chemical structure of a parallel T•AT triplet. Guerrini, L. and Alvarez-Puebla, R.A. Nanomaterials 2021, 11, 326. https://doi.org/10.3390/nano11020326. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

    The double stranded canonical helix (D1D2) consist of 31 base pairs in which strand D1 is pyrimidine rich and D2 is purine-rich strand (D2). A 22-nucleotide Triple helix forming oligonucleotide (TFO) that is rich in pyrimidines binds the 19 AT and 2 C-GC base triplets. The TFO binds along the major grove of the D2 strand which is purine rich.

    If the binding of the third strand in the major groove occurs at site where RNA polymerase binds to a gene, then third stand can inhibit gene transcription. Binding can also lead to a mutation or recombination at the site.

    Figure \(\PageIndex{28}\) below shows the base pairing of purine and pyrimidines of the third strand to the canonical AT dn GC base pairs of the original double-stranded DNA.

    triplehelicesBPs.svg

     

    Figure \(\PageIndex{28}\): Base pairing in triple helix motifs. (after Jain et al. Biochimie. 2008. doi: 10.1016/j.biochi.2008.02.011

    Figure \(\PageIndex{29}\) below shows an interactive iCn3D model of a solution conformation of a parallel DNA triple helix (1BWG).

    solution conformation of a parallel DNA triple helix (1BWG).png

    Figure \(\PageIndex{29}\): Solution conformation of a parallel DNA triple helix (1BWG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...5JU813eNjND8E7

    Triple helix formation can also occur within a single strand of DNA. The resulting structure is called H-DNA. An example is shown below. Note that the central blue, black and red sequences are all mirror image repeats (around a central nucleotide). During processes that unravel DNA (replication, transcription, repair), self-association of individual mirror repeats can form a locally stable triple helix, as shown in Figure \(\PageIndex{30}\) below.

    A_tunable_assay_for_modulators_of_genome-destabili_TripHelixFig1.svg

    Figure \(\PageIndex{30}\): Schematic illustrations of (A) the H-DNA or intramolecular triplex structure used in this study;del Mundo et al. (2019) Nucleic acids research. 47. e73. 10.1093/nar/gkz237. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/)

    The * between in the G*G and A*A denote Hoogsteen hydrogen bonding (purine motifs) in this intramolecular triple helix. Reverse Hoogsteen hydrogen bonds can also occur.

    Triple helices can form when single stranded DNA formed during replication, transcription or DNA repair with half of the required mirror symmetry folds back into the adjacent major grove and base pairs using Hoogsteen/reverse Hoogsteen bonding, which can be stabilized by Mg2+.

    Stability of nucleic acids

    After looking at the myriad of structures showing the nearly parallel hydrogen bonded base pairs, and from ideas from most textbooks and classes you have taken, you probably think that double-stranded DNA is held together and stabilized by hydrogen bonds between the bases. It is well known that greater the percentage of GC compared to AT, the greater the stability of the dsDNA, which translates into a higher "melting temperature (TM)", the temperature at which the dsDNA is converted to ssDNA. In fact, there is a linear relationship between GC content and TM. The figures above show that GC base pairs have 3 interbase hydrogen bonds compared to 2 in AT base pairs. This observations support the simple notion that interbase hydrogen bonds is the source of dsDNA stability.

    You would be in general correct in this belief, but you'd be missing the more important contributor to ds-DNA stability, base (π) stacking and the noncovalent interactions associated with the stacking. The main contributors to stability are hydrophobic interactions in the anhydrous hydrogen-bonded base pairs in the helix. Given that the hydrogen bond donors and acceptors that contribute to base pairing exist in the absence of competing water, the donors and acceptors are free to fully engage in bonding. The hydrogen bond interaction energy is hence more favorable in the stack. The stacking energy has been shown to be similar for an AT - AT stack and a GC-GC stack (about -9.8 kcal/mol, 41 kJ/mol). Hence AT and GC base pairs contribute equally to stability. The excess stability of dsDNA enriched in GC base pairs can still be explained by the extra stabilization for an additional hydrogen bond per GC base pair

    Proteins are stabilized by a myriad of interactions, but the folded state is marginally more stable than the ensemble of unfolded state. Marginal stability is important as protein conformation often must be perturbed on binding and ensuing function. The same must be true of double-stranded DNA, which must "unfold' or separate on replication, transcription and repair. It is well know that dsDNA structure is sensitive to hydration (see section on A, B, and Z DNA). Small molecules like urea, as we saw with proteins, can also denature DNA into single strands.

    DNA must be stabile enough to be the carrier of genetic information but dynamic enough to allow events that required partial unfolding. Others water-soluble molecules like ethylene glycol ethers (polyethylene glycol-400) and diglyme (dimethyl ether of diethylene glycol), which are more hydrophobic than water, appear to reduce base stacking interactions while maintaining them, and at the same time allow longitudinal extension or breathing of the helix. This dynamic extension may be required for transitions of B-DNA to Z-DNA, for example. The extensions also allows transient "hole" to appear between base pairs which might assist in the binding of intercolating agents like some transition metal complexes. The extension caused by these ethers and natural extensions would decrease base stacking but appear at the same time strengthening the hydrogen bonding between bases.

    Longitudinal helical extensions might be important when homologous gene recombine. In that process the homologous DNA strand but exchange with a paired homolog. This processing is associated with strand extension and disruption of base pair at every third base. Recombination also must allow chain extension as it maintains base-pairing fidelity.

    DNA structures gets obviously more complicated as it packs into the nucleus of a cell and forms chromosomes, as shown in Figure \(\PageIndex{31}\) below. We will study packing of DNA in other sections.

     

    DNAtoChromosome.svgFigure \(\PageIndex{31}\): Packing of DNA into chromosome. https://commons.wikimedia.org/wiki/F...omosome_en.svg. Creative Commons Attribution 3.0 Unported

    References

    Börner, R., Kowerko, D., Miserachs, H.G., Shaffer, M., and Sigel, R.K.O. (2016) Metal ion induced heterogeneity in RNA folding studied by smFRET. Coordination Chemistry Reviews 327 DOI: 10.1016/j.ccr.2016.06.002 Available at: https://www.researchgate.net/publication/303846502_Metal_ion_induced_heterogeneity_in_RNA_folding_studied_by_smFRET

    Hardison, R. (2019) B-Form, A-Form, and Z-Form of DNA. Chapter in: R. Hardison’s Working with Molecular Genetics. Published by LibreTexts. Available at: https://bio.libretexts.org/Bookshelves/Genetics/Book%3A_Working_with_Molecular_Genetics_(Hardison)/Unit_I%3A_Genes%2C_Nucleic_Acids%2C_Genomes_and_Chromosomes/2%3A_Structures_of_Nucleic_Acids/2.5%3A_B-Form%2C_A-Form%2C_and_Z-Form_of_DNA

    Lenglet, G., David-Cordonnier, M-H., (2010) DNA-destabilizing agents as an alternative approach for targeting DNA: Mechanisms of action and cellular consequences. Journal of Nucleic Acids 2010, Article ID: 290935, DOI: 10.4061/2010/290935 Available at: https://www.hindawi.com/journals/jna/2010/290935/

    Mechanobiology Institute (2018) What are chromosomes and chromosome territories? Produced by the National University of Singapore. Available at: https://www.mechanobio.info/genome-regulation/what-are-chromosomes-and-chromosome-territories/

    National Human Genome Research Institute (2019) The Human Genome Project. National Institutes of Health. Available at: https://www.genome.gov/human-genome-project

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    This page titled 8.1: Nucleic Acids - Structure and Function is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.

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