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.     

A molecular orbital study of stability and the conformation of double-stranded DNA-like polymers

The stacking and hydrogen bonding energies between bases in the B form of DNA were calculated by a perturbation method using the wave functions by the CNDO and the P-P-P methods. The exchange energies were calculated by using the corresponding orbitals. The magnitudes of the sums of the average stacking and hydrogen bonding energies per base pair of double-stranded DNA-like polymers are in good parallel with the melting temperatures of the polymers. The polymers containing I-C pairs are exceptions to this relation. Intrastrand stacking bases have the potential minimum at the distances of 2·8–3·7 Å. The minimum of stacking energy of double-stranded polymer for rotation of base pair around the helix axis exists near 36°. The deviation of the potential minimum from 36° seems to parallel the feature of the X-ray diffraction pattern of the polymer.     

Theoretical calculations of base-base interactions in nucleic acids: II. Stacking interactions in polynucleotides.

Base-base interactions were computed for single- and double stranded poly,ucleotides, for all possible base sequences. In each case, both right and left stacking arrangements are energetically possible. The preference of one over the other depends upon the base-sequence and the orientation of the bases with respect to helix-axis. Inverted stacking arrangement is also energetically possible for both single- and double-stranded polynucleotides. Finally, interacting energies of a regular duplex and the alternative structures were compared. It was found that the type II model is energetically more favourable than the rest.     

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.     

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 a full turn of an A-DNA duplex d(CGCGGGTACCCGCG)2

We report the 2.6 Å resolution crystal structure of the tetra-decamer d(CGCGGGTACCCGCG) in the tetragonal space group P4₃. This sequence contains the KpnI restriction site GGTACC in the centre which is flanked by alternating ‘CG’ sequences, and has a ‘TA’ step at the centre. These are features could favour the left-handed Z type helix. Despite this, overall the molecule has the A form. This is the first tetra-decamer crystallized in the A-DNA conformation, i.e. more than one full turn of the A helix. The crystallographic asymmetric unit consists of one tetra-decamer duplex. The helical twist and slide, as well as the base pair–base pair stacking interactions show alternations at the alternating pyrimidine–purine and purine–pyrimidine base steps. This variation is reminiscent of the dinucleotide repeat in left-handed Z-DNA helices. The crystal packing is unlike other A-DNA crystal structures, with each helix having a large number of contacts of many different types with symmetry-related neighbours.     

Prediction of hybridization and melting for double-stranded nucleic acids

This article presents a general statistical mechanical approach to describe self-folding together with the hybridization between a pair of finite length DNA or RNA molecules. The model takes into account the entire ensemble of single- and double-stranded species in solution and their mole fractions at different temperatures. The folding and hybridization models deal with matched pairs, mismatches, symmetric and asymmetric interior loops, bulges, and single-base stacking that might exist at duplex ends or at the ends of helices. All possible conformations of the single- and double-stranded species are explored. Only intermolecular basepairs are considered in duplexes at this stage.In particular we focus on the role of stacking between neighboring nucleotide residues of single unfolded strands as an important source of enthalpy change on helix formation which has not been modeled computationally thus far. Changes in the states of the single strands with temperature are shown to lead to a larger heat effect at higher temperature. An important consequence of this is that predictions of enthalpies, which are based on databases of nearest-neighbor energy parameters determined for molecules or duplexes with lower melting temperatures compared with the melting temperatures of the oligos for which they are used as a predictive tool, will be underestimated.     

Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.     

Cooperative nonenzymic base recognition II. thermodynamics of the helix-coil transition of oligoadenylic + oligouridylic acids

The properties of oligonucleotide helices of adeuylic- and uridylic acid oligomers have been investigated by measurements of hypo-and hyperchromieity. High ionic strengths favor the formation of triple helices. Thus, the double helix-coil transition can be studied (without interference by triple helices) only at low ionic-strength. A “phase diagram” is given representing the T_m-values of the various transitions at different ionic strengths for the system A(pA)₁₇ + U(pU)₁₇. Oligonucleolides of chain lengths <8 always form both double and triple helices at the nucleotide concentrations required for base pairing. For this reason the double helix-coil transition without coupling of the triple helix equilibrium can only be measured for chain lengths higher than 7. Melting curves corresponding to this transition have been determined for chain lengths 8, 9, 10, 11, 14 and 18 at different concentrations. An increase in nucleotide concentration leads to an increase in melting temperature. The shorter the chain length the lower the T_m-value and the broader the helix-coil transition. The experimental transition curves have been analysed according to a staggering zipper model with consideration of the stacking of the adeuylic acid single strands and the electrostatic repulsion of tlip phosphate charges on opposite strands. The temperature dependence of the nucleation parameter has been accounted for by a slacking factor x. The stacking factor expresses the magnitude of the stacking enthalpy. By curve fitting xwas computed to be 0.7, corresponding to a stacking enthalpy of about S kcal/mole. The model described allows the reproduction of the experimental transition curves with relatively high accuracy. In an appendix the thermodynamic parameters of the stacking equilibrium of poly A and of the helix-coil equilibria of poly A + poly U at neutral pH are calculated (ΔH_A = ?7.9 kcal/mole for the poly A stacking and ΔH₁₂ = ?10.9 kcal/mole for the formation of the double helix from the randomly coiled single strands). A formula for the configurational entropy of polymers derived by Flory on the basis of a liquid lattice model is adapted to calculate the stacking entropies of adenylic oligomers.     

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.     

Intramolecular triple helix as a model for regular polyribonucleotide (CAA)n

The regular (CAA)_n polyribonucleotide, as well as the omega leader sequence containing (CAA)-rich core, have recently been shown to form cooperatively melted and compact structures. In this report, we propose a structural model for the (CAA)_n sequence in which the polyribonucleotide chain is folded upon itself, so that it forms an intramolecular triple helix. The triple helix is stabilized by hydrogen bonding between bases thus forming coplanar triads, and by stacking interactions between the base triads. A distinctive feature of the proposed triple helix is that it does not contain the canonical double-helix elements. The difference from the known triple helices is that Watson-Crick hydrogen bond pairings do not take place in the interactions between the bases within the base triads.     

Sequence-dependent molecular conformation of polynucleotides: right and left-handed helices

Based upon a stereochemical guideline, two topologically distinct types of helicalduplexes have been deduced for a polynucleotide duplex with alternating purine pyrimidine sequence (PAPP): (a) right-handed uniform (RU) helix and (b) left-handed zig-zag (LZ) helix. Both structures have trinucleoside diphosphate as the basic unit wherein the purine pyrimidine fragment has a different conformation from the pyrimidine-purine fragment. Thus, RU and LZ helices represent two different classes of sequence-dependent molecular conformations for PAPP. The conformationalf eatures of an RU helix of PAPP in B-form and three LZ-helices for B-, D- and Z-forms are discussed.     

Fibre X-ray study of the helix-helix transitions of poly(dG-dC).poly(dG-dC).

Poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) present helix-helix transitions which are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2')-endo and C(3')-endo sugar puckering. Dihedral angles, sets of atomic co-ordinates and stereo views of the two S-DNA structures are given together with curves of calculated diffracted intensities.     

The influence of a chiral amino acid on the helical handedness of PNA in solution and in crystals

The X-ray structure of a self-complementary PNA hexamer (H-CGTACG-L-Lys-NH(2)) has been determined to 2.35 A resolution. The introduction of an L-lysine moiety has previously been shown to induce a preferred left-handedness of the PNA double helices in aqueous solution. However, in the crystal structure an equal amount of interchanging right- and left-handed helices is observed. The lysine moieties are pointing into large solvent channels and no significant interactions between this moiety and the remaining PNA molecule are observed. In contrast, molecular mechanics calculations show a preference for the left-handed helix of this hexameric PNA in aqueous solution as expected. The calculations indicate that the difference in the free energy of solvation between the left-handed and the right-handed helix is the determining factor for the preference of the left-handed helix in aqueous solution.     

The lattice energetics of some polypeptide chains

The interchain energetics of alpha, beta, and PGII conformations of polyglycine, the PPII and left-handed 3.30 fold helical conformations of trans poly-L -proline, and the Yonath and Traub triple helix and left-handed three fold helices of poly(gly-pro-pro) were investigated. Intra- and inter-chain stabilization energies appear to be inversely related, and the interchain stabilization energy can be as large its the intrachain energy. The minimization of the interchain energy can be described by the simultaneous optimization of interchain hydrogen bonding and intermolecular-sidechain digitation. The stability of the poly(gly-pro-pro) triple helix can be readily explained in terms of these two factors. In all cases the experimentally observed lattice packing is predicted, although the calculated lattice constants are slightly larger than those observed. The small differences between observed and predicted lattice constants probably reflect small errors in present conformational potential functions. Homopolymers are probably the best systems to use in the refinement of conformational potential functions because solvent effects arc minimized and the experimentally observed lattice constants provide a check on the configurational calculations.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

On the free-energy changes in the synthesis and degradation of nucleic acids.

Standard free-energy changes for reactions involving single- and double-stranded nucleic acids have been related to that for polynucleotide synthesis from ribonucleoside diphosphates for which deltaG degrees' approximately O. For polynucleotide formation from triphosphates this quantity is about -1 kcal. In the replication reaction the base pairing interactions are quantitatively of comparable importance. Production of a hydrolytic break in a double strand is substantially less favorable than in a single strand. The resealing of breaks utilizing ATP and NAD+ have similar free-energy changes and are entropy driven processes. The highly exergonic hydrolysis of pyrophosphate is maintained to be of significance for both in vivo and in vitro polymerizations.     

Hybrid hexanucleotide duplex containing cyclonucleotides and deoxynucleotides: the d(TA) segment can adopt a high anti left-handed double-helical structure

It is known that oligonucleotides containing cyclonucleosides with a high anti (intermediate between anti and syn) glycosidic conformation adopt left-handed, single- and double-helical structures [Uesugi, S., Yano, J., Yano, E., & Ikehara, M. (1977) J. Am. Chem. Soc. 99, 2313-2323]. In order to see whether DNA can adopt the high anti left-handed double-helical structure or not, a self-complementary hexanucleotide containing 6,2'-O-cyclocytidine (C(o)), 8,2'-O-cycloguanosine (G(o)), thymidine, and deoxyadenosine, C(o)G(o)dTdAC(o)G(o), was synthesized. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C(o)G(o)dTdAC(o)G(o) adopts a left-handed double-helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. A conformational model of the left-handed duplex was obtained by calculation with energy minimization. Thus it appears that DNA can form a high anti, left-handed double helix under some constrained conditions, which is quite different from that of Z-DNA.