The dimensions and shapes of the furanose rings in nucleic acids

A survey was made of the geometry of furanose rings in beta-nucleotides and beta-nucleosides (as monomers related to nucleic acids) for which structures have been determined by X-ray crystallography. Mean values, and estimated standard deviations from them, were calculated for bond-lengths, bond-angles and conformation-angles. For parameters with values dependent on ring-puckering, separate calculations were made for each ring type. (The rings are puckered in one of three conformations: C-2- or C-3-endo or C-3-exo; C-2-exo has not been observed.) The results were used to compute standard furanose rings with C-2-endo, C-3-endo and C-3-exo conformations for use in nucleic acid molecular model-building. The survey also showed that the only other conformation-angle in nucleotides dependent on the furanose ring conformation corresponds to the relative orientation of the purine (or pyrimidine) base and the ring.     

Crystal structures of B-DNA with incorporated 2'-deoxy-2'-fluoro-arabino-furanosyl thymines: implications of conformational preorganization for duplex stability.

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.     

Backbone-base inclination as a fundamental determinant of nucleic acid self- and cross-pairing

The crystal structure of the duplex formed by oligo(2',3'-dideoxy-beta-d-glucopyranosyl)nucleotides (homo-DNA) revealed strongly inclined backbone and base-pair axes [Egli,M., Pallan,P.S., Pattanayek,R., Wilds,C.J., Lubini,P., Minasov,G., Dobler,M., Leumann,C.J. and Eschenmoser,A. (2006) Crystal structure of homo-DNA and nature's choice of pentose over hexose in the genetic system. J. Am. Chem. Soc., 128, 10847-10856]. This inclination is easily perceived because homo-DNA exhibits only a modest helical twist. Conversely, the tight coiling of strands conceals that the backbone-base inclinations for A- (DNA and RNA) and B-form (DNA) duplexes differ considerably. We have defined a parameter eta(B) that corresponds to the local inclination between sugar-phosphate backbone and base plane in nucleic acid strands. Here, we show its biological significance as a predictive measure for the relative strand polarities (antiparallel, aps, or parallel, ps) in duplexes of DNA, RNA and artificial nucleic acid pairing systems. The potential of formation of ps duplexes between complementary 16-mers with eight A and U(T) residues each was investigated with DNA, RNA, 2'-O-methylated RNA, homo-DNA and p-RNA, the ribopyranosyl isomer of RNA. The thermodynamic stabilities of the corresponding aps duplexes were also measured. As shown previously, DNA is capable of forming both ps and aps duplexes. However, all other tested systems are unable to form stable ps duplexes with reverse Watson-Crick (rWC) base pairs. This observation illustrates the handicap encountered by nucleic acid systems with inclinations eta(B) that differ significantly from 0 degrees to form a ps rWC paired duplex. Accordingly, RNA with a backbone-base inclination of -30 degrees , pairs strictly in an aps fashion. On the other hand, the more or less perpendicular orientation of backbone and bases in DNA allows it to adopt a ps rWC paired duplex. In addition to providing a rationalization of relative strand polarity with nucleic acids, the backbone-base inclination parameter is also a determinant of cross-pairing. Thus, systems with strongly deviating eta(B) angles will not pair with each other. Nucleic acid pairing systems with significant backbone-base inclinations can also be expected to display different stabilities depending on which terminus carries unpaired nucleotides. The negative inclination of RNA is consistent with the higher stability of duplexes with 3'- compared to those with 5'-dangling ends.     

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.     

Volumetric properties of nucleic acids

Volumetric studies can yield useful new information on a myriad of intra- and intermolecular interactions that stabilize nucleic acid structures. In particular, appropriately designed volumetric measurements can characterize the conformation-dependent hydration properties of nucleic acids as a function of solution conditions, including temperature, pressure, ionic strength, pH, and cosolvent concentration. We have started to accumulate a substantial database on volumetric properties of DNA and RNA, as well as on related low molecular weight model compounds. This database already has provided unique insights into the molecular origins of various nucleic acid recognition processes, including helix-to-coil and helix-to-helix conformational transitions, as well as drug-DNA interactions. In this article, we review recent progress in volumetric investigations of nucleic acids, emphasizing how these data can be used to gain insight into intra-and intermolecular interactions, including hydration properties. Throughout this review, we underscore the importance of volume and compressibility data for characterizing the hydration properties of nucleic acids and their constituents. We also describe how such volumetric data can be interpreted at the molecular level to yield a better understanding of the role that hydration can play in modulating the stability and recognition of nucleic acids.     

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.     

2'-Fluoroarabino- and arabinonucleic acid show different conformations, resulting in deviating RNA affinities and processing of their heteroduplexes with RNA by RNase H

2'-Deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA) paired to RNA are substrates of RNase H. The conformation of the natural DNA/RNA hybrid substrates appears to be neither A-form nor B-form. Consistent with this, the conformations of FANA and ANA were found to be intermediate between the A- and B-forms. However, FANA opposite RNA is preferred by RNase H over ANA, and the RNA affinity of FANA considerably exceeds that of ANA. By investigating the conformational boundaries of FANA and ANA residues in crystal structures of A- and B-form DNA duplexes at atomic resolution, we demonstrate that FANA and ANA display subtle conformational differences. The structural data provide insight into the structural requirements at the catalytic site of RNase H. They also allow conclusions with regard to the relative importance of stereoelectronic effects and hydration as modulators of RNA affinity.     

FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.     

Effect of LNA modifications on small molecule binding to nucleic acids

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.     

Why ribose was selected as the sugar component of nucleic acids

During evolution ribose was selected as the exclusive sugar component of nucleic acids. The selection is explained by using molecular models and by eliminating most of the other common sugars by looking at their chemical structure and envisioning how they would fit in a nucleic acid model. Comparisons of sugar pucker conformations and configurations of pentoses indicate that ribose was not randomly selected but the only choice, since beta-D-ribose fits best into the structure of physiological forms of nucleic acids. In other nucleotides containing arabinose, xylose, or lyxose, the C(2)'-OH and/or the C(3)'-OH are above the furanose ring, causing steric interference with the bulky base and the C(5)'-OH group.     

Modeling nucleic acids

Nucleic acids are an important class of biological macromolecules that carry out a variety of cellular roles. For many functions, naturally occurring DNA and RNA molecules need to fold into precise three-dimensional structures. Due to their self-assembling characteristics, nucleic acids have also been widely studied in the field of nanotechnology, and a diverse range of intricate three-dimensional nanostructures have been designed and synthesized. Different physical terms such as base-pairing and stacking interactions, tertiary contacts, electrostatic interactions and entropy all affect nucleic acid folding and structure. Here we review general computational approaches developed to model nucleic acid systems. We focus on four key areas of nucleic acid modeling: molecular representation, potential energy function, degrees of freedom and sampling algorithm. Appropriate choices in each of these key areas in nucleic acid modeling can effectively combine to aid interpretation of experimental data and facilitate prediction of nucleic acid structure.     

NMR structure of a DNA duplex containing nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the unmodified control

The three-dimensional structures of two DNA duplexes d(CATGAGTAC). d(GTACXCATG) (1) and d(CATGAGTAC).d(GTACTCATG) (2), where X represents 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, were solved using high resolution nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Good convergence was observed between final structures derived from A- and B-form starting geometries for both 1 and 2. Structures of 1 and 2 are right-handed duplexes within the B-form conformational regime. Furthermore, the structures of 1 and 2 are highly similar, with differences in the structures localized to the vicinity of residue 14 (X versus T). The pyrrole group of 1 is in the syn conformation and it is displaced towards the major groove. Furthermore, unlike T14 in 2, the base of X14 has reduced pi-pi stacking interactions with C13 and C15 and the nitro group of X14 is pointing out of the major groove. The structures presented here establish the basis of the thermal data of DNA duplexes containing X and should be informative during the design of improved wild card nucleobase analogs.     

DNA-RNA hybrid secondary structures

DNA-RNA and DNA-DNA duplexes are even more polymorphic than observed previously. DNA-RNA hybrids can have secondary structures like A-DNA or A-RNA, but double helices of the synthetic DNA-RNA hybrids poly(dA) X poly(rU) and poly(dI) X poly(rC), respectively, form 11-fold and 10-fold double-helical structures in which the two chains have quite different conformations. Extensive X-ray fiber diffraction analyses show that in both structures the DNA chains have C-2'-endo-puckered furanose rings, while the anti-parallel RNA chains have C-3'-endo-puckered rings. The bidirectional properties of such duplexes may be important in the transfer of biological information from nucleic acids.     

Structural basis for the unusual properties of 2',5' nucleic acids and their complexes with RNA and DNA

To provide insights into the unusual properties of 2',5' nucleic acids (iso nucleic acids), that includes their rejection by Nature as information molecules, modeling studies have been carried out to examine if they indeed possess the stereochemical ability to form helical duplexes and triplexes, just as their 3',5' linked constitutional isomers. The results show that the formation of helical duplexes with 2',5' linkages demands a mandatory displacement of the Watson and Crick base pairs from the helical axis, as a direct consequence of the lateral shift of the sugar-phosphate backbone from the periphery towards the interior of the helix. Thus, both duplexes and triplexes formed with a 2',5'-sugar-phosphate backbone possess this intrinsic trait, manifested normally only in A type duplexes of DNA and RNA. It was found that only a 10-fold symmetric parallel triplex with isomorphous T.AT triplets is stereochemically favorable for isoDNA with 'extended' nucleotide repeats, unlike the 12-fold symmetric triplex favored by DNA. The wider nature of a 12-fold triplex, concomitant with mandatory slide requirement for helix formation in isoDNA, demands even larger displacement, especially with 'extended' nucleotide structural repeats, thereby violating symmetry. However, a symmetric triplex possessing higher twist, can be naturally formed for isoDNA with a 'compact' nucleotide repeat. Two nanosecond molecular dynamics simulation of a 2',5'-B DNA duplex, formed with an intrinsic base pair displacement of -3.3 A, does not seem to favor a total transition to a typical A type duplex, although enhanced slide, X-displacement, decrease in helical rise and narrowing of the major groove during simulation seem to indicate a trend. Modeling of the interaction between the chimeric isoDNA.RNA duplex and E. coli RNase H has provided a structural basis for the inhibitory action of the enzyme. Interaction of residues Gln 80, Trp 81, Asn 16 and Lys 99, of E. coli RNase H with DNA of the DNA.RNA hybrid, are lost when the DNA backbone is replaced by isoDNA. Based on modeling and experimental observations, it is argued that 2',5' nucleic acids possess restricted conformational flexibility for helical polymorphism. The inability of isoDNA to favor the biologically relevant B form duplex and the associated topological inadequacies related to nucleic acid compaction and interactions with regulatory proteins may be some of the factors that might have led to the rejection of 2',5' links.     

Conformational studies of nucleic acids. I. A rapid and direct method for generating furanose coordinates from the pseudorotation angle

The greatest difficulty in modeling a nucleic acid is generating the coordinates of its furanoses. This difficulty arises from constraints imposed by the closed ring geometries of these sugars. We have developed a new method for modeling these furanose rings. Using this method, the coordinates of a sugar can be obtained quickly and unambiguously for any point on the pseudorotational pathway from one parameter: the phase angle of pseudorotation P. The significant difference between this and previous sugar modeling schemes is that here the endocyclic bond lengths of the five-membered sugar ring are allowed to vary a small amount according to simple, explicit, and experimentally reasonable analytic functions of P. The coefficients of these functions follow from the empirical behavior of the endocyclic bond angles and from geometrical constraints due to ring closure. The ability to model the sugars directly from one parameter greatly facilitates carrying out the global conformational studies on nucleic acid constituents which will be attempted in subsequent papers of this series.     

Conformational studies of nucleic acids: IV. The conformational energetics of oligonucleotides: d(ApApApA) and ApApApA

Utilizing a new method for modeling furanose pseudorotation (D. A. Pearlman and S.-H. Kim, J. Biomol. Struct. Dyn. 3, 85 (1985)) and the empirical multiple correlations between nucleic acid torsion angles we derived in the previous report (D. A. Pearlman and S.-H. Kim, previous paper in this issue), we have made an energetic examination of the entire conformational spaces available to two nucleic acid oligonucleotides: d(ApApApA) and ApApApA. The energies are calculated using a semi-empirical potential function. From the resulting body of data, energy contour map pairs (one for the DNA molecule, one for the RNA structure) have been created for each of the 21 possible torsion angle pairs in a nucleotide repeating unit. Of the 21 pairs, 15 have not been reported previously. The contour plots are different from those made earlier in that for each point in a particular angle-angle plot, the remaining five variable torsion angles are rotated to the values which give a minimum energy at this point. The contour maps are overall quite consistent with the experimental distribution of oligonucleotide data. A number of these maps are of particular interest: delta (C5'-C4'-C3'-O3')-chi (O4'-C1'-N9-C4), where the energetic basis for an approximately linear delta-chi correlation can be seen: zeta (C3'-O3'-P-O5')-delta, in which the experimentally observed linear correlation between zeta and delta in DNA(220 degrees less than zeta less than 280 degrees) is clearly predicted; zeta-epsilon (C4'-C3'-O3'-P), which shows that epsilon increases with decreasing zeta less than 260 degrees; alpha (O3'-P-O5'-C5')-gamma (O5'-C5'-C4'-C3') where a clear linear correlation between these angles is also apparent, consistent with experiment; and several others. For the DNA molecule studied here, the sugar torsion delta is predicted to be the most flexible, while for the RNA molecule, the greatest amount of flexibility is expected to reside in alpha and gamma. Both the DNA and RNA molecules are predicted to be highly polymorphic. Complete energy minimization has been performed on each of the minima found in the energy searches and the results further support this prediction. Possible pathways for B-form to A-form DNA interconversion suggested by the results of this study are discussed. The results of these calculations support use of the new sugar modeling technique and torsion angle correlations in future conformational studies of nucleic acids.     

Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.     

Vaccinia virus RNA helicase: nucleic acid specificity in duplex unwinding.

Vaccinia virus RNA helicase (NPH-II) catalyzes nucleoside triphosphate-dependent unwinding of duplex RNAs containing a single-stranded 3' RNA tail. In this study, we examine the structural features of the nucleic acid substrate that are important for helicase activity. Strand displacement was affected by the length of the 3' tail. Whereas NPH-II efficiently unwound double-stranded RNA substrates with 19- or 11-nucleotide (nt) 3' tails, shortening the 3' tail to 4 nt reduced unwinding by an order of magnitude. Processivity of the helicase was inferred from its ability to unwind a tailed RNA substrate containing a 96-bp duplex region. NPH-II exhibited profound asymmetry in displacing hybrid duplexes composed of DNA and RNA strands. A 34-bp RNA-DNA hybrid with a 19-nt 3' RNA tail was unwound catalytically, whereas a 34-bp DNA-RNA hybrid containing a 19-nt 3' DNA tail was 2 orders of magnitude less effective as a helicase substrate. NPH-II was incapable of displacing a 34-bp double-stranded DNA substrate of identical sequence. 3'-Tailed DNA molecules with 24- or 19-bp duplex regions were also inert as helicase substrates. On the basis of current models for RNA-DNA hybrid structures, we suggest the following explanation for these findings. (i) Unwinding of duplex nucleic acids by NPH-II is optimal when the polynucleotide strand of the duplex along which the enzyme translocates has adopted an A-form secondary structure, and (ii) a B-form secondary structure impedes protein translocation through DNA duplexes.