The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.

Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed.     

Conformational studies of (2''-5'') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.     

Spatial configuration of ordered polynucleotide chains. V. Conformational energy estimates of helical structure

Semiempirical potential energy functional used previously to account successfully for the mean-square unperturbed dimensions and nmr coupling constants of randomly coiling polynucleotides are used, after modifications, to account for base stacking and interstrand hydrogen bonding, and to evaluate the conformational energies of single- and double-stranded polynucleotide helices. Attention is focused upon the variety of A-genus helices with local backbone conformations resembling the known double-helical structures of RNA. Distinct structural differences between single- and double-stranded helices are predicted from the energy calculations. A second point of interest is the apparent failure of two conformationally identical left-handed polynucleotide chains to form a left-handed duplex. The third major observation of the study is the wide morphological variety of theoreticaly allowed right-handed polynucleotide duplexes. In addition to the familiar double helix stabilized by horizontal base stacking and hydrogen bonding, an unusual vertical double helix is predicted to form between complementary bases fixed in the unusual but not energetically forbidden high anti glycosyl conformation. Experimental results bearing upon the theoretical predictions are discussed.     

Analysis of the possible helical structures of nucleic acids and polynucleotides. Application of (n-h) plots.

The two helical parameters n and h where n is the number of nucleotide residues per turn and h is the height per nucleotide residue have been evaluated for single stranded helical polynucleotide chains comprising C(3') -endo and C(2') endo class of nucleotides. The helical parameters are found to be especially sensitive to the C(4')-C(3') (sugar pucker) and the C(4')-C(5') torsions. The (n-h) plots display only one important helix forming domain for each class of nucleotides characterized by the sugar pucker and the C(4')-C(5') torsion. A correlation between the (n-h) plots and the known RNA (A,A') and DNA (A,B,C) helical forms has been established. It is found that all forms of helices except the C-DNA possess a favorable combination of P-O torsions. The analysis of the (n-h) plots suggests that C-DNA can have a conformation very similar to B-DNA. Although the (n-h) plots predict the stereochemical possibility of both right-handed and left-handed helices, nucleic acids apparently prefer right-handed conformation because of the energetics associated with the sugar-phosphate backbone and the base.     

A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

Visualization of planar drug intercalations in B-DNA.

A computerized linked-atom modeling system was developed to examine the stereochemical requirements for intercalation of planar drugs into DNA. All classes of conformational possibilities for extending the polynucleotide backbone were examined for their ability to accommodate insertion of a drug into a base-paired region of DNA compatible with adjacent regions of B-DNA while stacking interactions, steric strain and non-bonded interatomic contacts were optimised. One conformation was found which proved superior to all others in ability to satisfy these criteria: an extension of the backbone by characteristic changes in two torsion angles to trans values, plus a change in one sugar puckering to C3'-endo to relieve strain in an adjacent residue. The turn angle distributed over three polynucleotides for this most general mode of intercalation is 90 degrees, equivalent to a helical unwinding of -18 degrees for B-DNA.     

Conformational dependence of the Raman scattering intensities from polynucleotides. 3. Order-disorder changes in helical structures

Raman spectra are presented on ordered and presumably helical structures of DNA and RNA as well as the poly A·poly U helical complex, polydAT, and the helical aggregates of 5′-GMP and 3′-GMP. The changes in the frequency and the intensity of the Raman bands as these structures undergo order-disorder transitions have been measured. In general the changes we have found can be placed into three categories: (1) A reduction in the intensities of certain ring vibrations of the polynucleotide bases is observed when stacking or ordering occurs (Raman hypochromism). Since the ring vibrational frequencies are different for each type of base, we have been able to obtain some estimate of average amount of order of each type of base in partially ordered helical systems. (2) A very large increase in the intensity of a sharp, strongly polarized band at about 815 cm^?1 is observed when polyriboA and polyriboU are formed into a helical complex. Although this band is not present in the separated chains at high temperature, a broad diffuse band at about 800 cm^?1 is present. The 815 cm^?1 band undoubtedly arises from the vibrations of the phosphate-sugar portions of the molecule and provides a sensitive handle to the back-bone conformation of the polymer. This band also appears upon ordering of RNA, formation of the helical aggregate of 5′-riboGMP, and to some extent in the selfstacking of the polyribonucleotides polyA, polyU in the presence of Mg⁺⁺, PolyC, and polyG. No such intense, polarized band is found, however, in ordered DNA, polydAT, or the 3′-riboGMP aggregate, although there is a conformationally independent band at about 795 cm^?1 in DNA and polydAT. (3) Numerous frequency changes occur during Conformational changes. In particular the 1600–1700 cm^?1 region in D₂O shows significant conformationally dependent changes in the C?O stretching region analogous to the changes in this region which have been observed in these substances in the infrared. Thus, Raman scattering appears to provide a technique for simultaneously observing the effects of base stacking, backbone conformation and carbonyl hydrogen bonding in nucleic acids in moderately dilute (10–25 mg/ml) aqueous solutions.     

Low temperature solution structures and base pair stacking of double helical d(CGTACG)(2)

Solution structures and base pair stacking of a self- complementary DNA hexamer d(CGTACG)(2) have been studied at 5, 10 and 15 degrees C, respectively. The stacking interactions among the center base pair steps of the DNA duplex are found to improve when the terminal base pairs became less stable due to end fraying. A new structural quantity, the stacking sum (Sigma(s)), is introduced to indicate small changes in the stacking overlaps between base pairs. The improvements in the stacking overlaps to maintain the double helical conformation are probably the cause for the observed temperature dependent structural changes in double helical DNA molecule. A detailed analysis of the helical parameters, backbone torsion angles, base orientations and sugar conformations of these structures has been performed.     

A Proposal For A Specific Double-Helical Structure In Which The Polynucleotide Strands Intercalate Instead of Forming Base-pairs

Abstract

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen- bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5′-dCMPNa₂ which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3Å. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30° and a base separation of 6.6Å along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5Å, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.     

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Backbone conformation in nucleic acids: an analysis of local helicity through heminucleotide scheme and a proposal for a unified conformational plot

A relationship has been established to express the local helicity of a polynucleotide backbone directly in terms of the virtual bonds spanning the conformationally equivalent heminucleotide repeats, with a view to provide a better understanding of the cumulative effects of all the chemical bond rotational variations on local helicity. Using this, an analysis made with a few oligodeoxynucleotide crystal structures clearly brings forth that it is the concerted movements manifested in the near neighbour correlations between the pair of chemical bonds C4'-C5' and P-O5' and C4'-C3' and P-O3' of the 5' and 3' heminucleotides respectively that are primarily responsible for the observed non-uniform helical twists both in A and B type helical backbones. That these need not be restricted to oligodeoxynucleotides but may be a feature of oligoribonucleotides backbone also is shown from an analysis of helical segments of yeast tRNA(Phe). A proposal of a unified or a grand two dimensional conformational plot which would help visualise succinctly the overall effect of the variations in all the repeating six chemical bonds of a polynucleotide backbone is made. Apart from considerable simplification, the plot affords identification on it regions characteristic of helical, and loop and bend conformations of nucleic acid backbone chain.     

UDP-glucose dehydrogenase: substrate binding stoichiometry and affinity

Precise structural parameters of polyribonucleotides single stranded helices are determined as well as those of double stranded helices of poly 2′-O-methyl A and of poly A at neutral and acid pH. Infrared linear dichroism investigations indicate the similarity of the conformation of the sugar-phosphate backbone of these single and double stranded helices. The angles of the phosphate group for single stranded helix at neutral pH is found to be oriented at 48° for the 02P02 bisector and at about 65° for the 02–03 line to the helix axis. Similar values were found for double stranded poly A helix at acid pH. These structural parameters obtained for the first time on single stranded polynucleotide helices are proposed to be valid for other similar helical chains such as poly A segments of nuclear or messenger RNA and single stranded CCA acceptor end of transfer RNA.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Backbone Conformation in Nucleic Acids: An Analysis of Local Helicity through Heminucleotide Scheme and a Proposal for a Unified Conformational Plot

Abstract

A relationship has been established to express the local helicity of a polynucleotide backbone directly in terms of the virtual bonds spanning the conformationally equivalent heminucleotide repeats, with a view to provide a better understanding of the cumulative effects of all the chemical bond rotational variations on local helicity. Using this, an analysis made with a few oligodeoxynucleotide crystal structures clearly brings forth that it is the concerted movements manifested in the near neighbour correlations between the pair of chemical bonds C4′—C5′ and P—05′ and C4′-C3′ and P-03′ of the 5′ and 3′ heminucleotides respectively that are primarily responsible for the observed non-uniform helical twists both in A and B type helical backbones. That these need not be restricted to oligodeoxynucleotides but may be a feature of oligoribonucleotides backbone also is shown from an analysis of helical segments of yeast tRNA^Phe. A proposal of a unified or a grand two dimensional conformational plot which would help visualise succinctly the overall effect of the variations in all the repeating six chemical bonds of a polynucleotide backbone is made. Apart from considerable simplification, the plot affords identification on it regions characteristic of helical, and loop and bend conformations of nucleic acid backbone chain.     

Conformations of dinucleoside monophosphates in relation to duplex DNA structures

Mononucleotide conformations are important in understanding the structural aspects of nucleic acids and polynucleotides. In order to study the influence of stacking interactions between adjacent bases in a polynucleotide on the preferred conformations of mononucleotides, conformational energy calculations have been carried out on dinucleoside monophosphate fragments. Four base sequences—d(ApT), d(TpA), d(CpG), and d(GpC)— have been analyzed in the framework of helical structures. Flexibility of the furanose ring has been incorporated in the investigations. Energetically favored conformers of the four compounds correspond to a variety of left- and right-handed uniform helical structures, similar to those of the commonly observed polymorphous forms. Implications of these investigations on the further understanding of double-helical polynucleotide conformations are briefly discussed.     

Application of Raman spectroscopy to biological macromolecules.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.     

Study of interaction of polynucleotide chains with oligomers by means of chromatography

A new method of measurement of the binding constant of oligonucleotides with complementary polynucleotide chains was developed using a gel filtration procedure. Data for complexing of poly U with triplets ApApA are presented and analyzed by means of a simple statistical theory of cooperative adsorption deduced by the Bethe-Guggenheim method. The complexes are triple, they contain two polymer chains bound by oligomers. The energy of stacking is found equal to 1300 cal/mol in fair agreement with earlier data of Ts'o. The energy of binding of a single triplet to both complementary chains is equal to 14,000 cal/mol.     

Energy hyperspace for stacking interaction in AU/AU dinucleotide step: Dispersion‐corrected density functional theory study

Double helical structures of DNA and RNA are mostly determined by base pair stacking interactions, which give them the base sequence‐directed features, such as small roll values for the purine–pyrimidine steps. Earlier attempts to characterize stacking interactions were mostly restricted to calculations on fiber diffraction geometries or optimized structure using ab initio calculations lacking variation in geometry to comment on rather unusual large roll values observed in AU/AU base pair step in crystal structures of RNA double helices. We have generated stacking energy hyperspace by modeling geometries with variations along the important degrees of freedom, roll, and slide, which were chosen via statistical analysis as maximally sequence dependent. Corresponding energy contours were constructed by several quantum chemical methods including dispersion corrections. This analysis established the most suitable methods for stacked base pair systems despite the limitation imparted by number of atom in a base pair step to employ very high level of theory. All the methods predict negative roll value and near‐zero slide to be most favorable for the purine–pyrimidine steps, in agreement with Calladine's steric clash based rule. Successive base pairs in RNA are always linked by sugar–phosphate backbone with C3′‐endo sugars and this demands C1′–C1′ distance of about 5.4 Å along the chains. Consideration of an energy penalty term for deviation of C1′–C1′ distance from the mean value, to the recent DFT‐D functionals, specifically ωB97X‐D appears to predict reliable energy contour for AU/AU step. Such distance‐based penalty improves energy contours for the other purine–pyrimidine sequences also. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 107–120, 2014.     

A molecular dynamics simulation of the (dG)6 . (dC)6 minihelix including counterions and water

The results of a 60 ps molecular dynamics (MD) simulation of (dG)6.(dC)6 including 10 Na+ counterions and 292 water molecules are presented. All backbone angles and helix parameters for the hexamer are reported in this paper along with trajectory plots of selected angles. Hydrogen bonding between the bases along the helical axis was observed to fluctuate with time, showing the dynamic nature of the base-pairing interaction. These fluctuations gave rise to unusual hydrogen-bonding patterns. Good intrastrand base stacking and no interstrand base stacking were also observed. The hexamer minihelix retains an essentially B-DNA conformation throughout the entire simulation even though some helix parameters and backbone angles do not have strict B-DNA values. The most striking feature obtained from the simulation was a high propeller twist, which resulted in a narrow minor groove for the minihelix. It is proposed that (dG)n.(dC)n sequences are resistant to DNAase I because of this narrow minor groove in dilute aqueous solution.