A New Strategy for DNA Inhibition with Backbone Substituted DNA Analogues

Abstract

Methylphosphotriester DNA shows a number of unique (bio)chemical properties: formation of parallel right-handed, anti-parallel left- and right-handed duplexes, and a high sequence-specific affinity for natural DNA. The impact of (pro)chirality of phosphorus in natural and modified DNA is discussed.     

The hydration of nucleic acid duplexes as assessed by a combination of volumetric and structural techniques

Using high precision densimetric and ultrasonic measurements, we have determined, at 25°C, the apparent molar volumes ΦV and the apparent molar compressibilities ΦK_S of four nucleic acid duplexes—namely, the DNA duplex, poly(dIdC)poly(dIdC); the RNA duplex, poly(rA)poly(rU); and the two DNA/RNA hybrid duplexes, poly(rA)poly(dT) and poly(dA)poly(rU). Using available fiber diffraction data on these duplexes, we have calculated the molecular volumes as well as the solvent‐accessible surface areas of the constituent charged, polar, and nonpolar atomic groups. We found that the hydration properties of these nucleic acid duplexes do not correlate with the extent and the chemical nature of the solvent‐exposed surfaces, thereby suggesting a more specific set of duplex–water interactions beyond general solvation effects. A comparative analysis of our volumetric data on the four duplexes, in conjunction with available structural information, suggests the following features of duplex hydration: (a) The four duplexes exhibit different degrees of hydration, in the order poly(dIdC)poly(dIdC) > poly(dGdC)poly(dGdC) > poly(dAdT)poly(dAdT) ≈ poly(dA)poly(dT). (b) Repetitive AT and IC sequences within a duplex are solvated beyond general effects by a spine of hydration in the minor groove, with this sequence‐specific water network involving about 8 additional water molecules from the second and, perhaps, even the third hydration layers. (c) Repetitive GC and IC sequences within a duplex are solvated beyond general effects by a “patch of hydration” in the major groove, with this water network involving about 13 additional water molecules from the second and, perhaps, even the third hydration layers. (d) Random sequence, polymeric DNA duplexes, which statistically lack extended regions of repetitive AT, GC, or IC sequences, do not experience such specific enhancements of hydration. Consequently, consistent with our previous observations (T. V. Chalikian, A. P. Sarvazyan, G. E. Plum, and K. J. Breslauer, Biochemistry, 1994, Vol. 33, pp. 2394–2401), duplexes with approximately 50% AT content exhibit the weakest hydration, while an increase or decrease from this AT content causes enhancement of hydration, either due to stronger hydration of the minor groove (an increase in AT content) or due to stronger hydration of the major groove (an increase in GC content). (e) In dilute aqueous solutions, a B‐DNA duplex is more hydrated than an A‐DNA duplex, a volumetric‐based conclusion that is in agreement with previous results obtained on crystals, fibers, and DNA solutions in organic solvent–water mixtures. (f) the A‐like, RNA duplex poly(rA)poly(rU) and the structurally similar A‐like, hybrid duplex poly(rA)poly(dT), exhibit similar hydration properties, while the structurally distinct A‐like, hybrid duplex poly(rA)poly(dT) and non‐A‐like, hybrid duplex poly(dA)poly(rU) exhibit differential hydration properties, consistent with structural features dictating hydration characteristics. We discuss how volumetric characterizations, in conjunction with structural studies, can be used to describe, define, and resolve the general and sequence/conformation‐specific hydration properties of nucleic acid duplexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 459–471, 1999     

Studies on Anticodon-anticodon Interactions: Hemi-protonation of Cytosines Induces Self-pairing Through the GCC Anticodon of E. Coli tRNA-Gly

Abstract

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemi- protonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.     

Conformational Study of DNA-RNA Duplexes Containing MMI Substituted Phosphodiester Linkages by FTIR Spectroscopy

Abstract

Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5′-GCGT*TT*TT*TT*TT*TGCG-3′) containing various combinations of 2′-O-methyl and 2′-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2′-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5′-CGCAAAAAAAAAACGC-3′) and the modified oligonucleotides a-f showed that all sugar residues adopted a C3′-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3′-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2′-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.     

Boranophosphate Nucleic Acids - A Versatile DNA Backbone

Abstract

Important chemical and biochemical properties of boranophosphate DNA and RNA oligonucleotides are reviewed. Stereoregular boranophosphate oligomers can be synthesized enzymatically and form stable duplexes with DNA. Fully boronated, non-stereoregular oligothymidylates, synthesized chemically, form hybrids with poly(A) that have lower melting points than oligothymidylate:poly(A), yet they nevertheless can support the RNase H mediated cleavage of RNA.     

Correlation of Tm,sequence, and ΔH of complementary RNA helices and comparison with DNA helices

T_m values of 16 fully complementary RNA duplexes with repeating base sequence have been employed as the empirical basis for developing a reliable and practical method for computing apparent enthalpies (ΔH _calc) for their helix → coil transitions. The approach taken is the same as in the accompanying investigation of DNA duplexes, although some of the computational variables of the “best-fit” function are necessarily different due to the distinguishing structural properties of the RNA-type helix. An excellent linear correlation was thus obtained between experimental T_m and ΔH _calc values. An equally good fit was obtained between T_m and ΔH _calc for five unrelated (to the 16 RNAs) decaribonucleotide duplexes. The differences in computational variables between the best-fit methods for RNA and DNA duplexes are shown to be a reflection of differences in cation binding and the effective local dielectric. The greater T_m dependence on G·C content of RNA helices than of DNA helices is shown to be due to a greater latitude of stacking stabilities of complementary dinucleotide fragments containing A·T than A·U base pairs.     

Alternation of DNA and Solvent Layers in the A Form of d(GGCGCC) Obtainhted by Ethanol Crystallization

Abstract

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)₂ in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2, 4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C222 ₁). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Abstract

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.     

More pronounced salt dependence and higher reactivity for platination of the hairpin r(CGCGUUGUUCGCG) compared with d(CGCGTTGTTCGCG)

The DNA interference pathways exhibited by cisplatin and related anticancer active metal complexes have been extensively studied. Much less is known to what extent RNA interaction pathways may operate in parallel, and perhaps contribute to both antineoplastic activity and toxicity. The present study was designed with the aim of comparing the reactivity of two model systems comprising RNA and DNA hairpins, r(CGCGUUGUUCGCG) and d(CGCGTTGTTCGCG), towards a series of platinum(II) complexes. Three platinum complexes were used as metallation reagents; cis-[PtCl(NH₃)₂(OH₂)]⁺ (1), cis-[PtCl(NH₃)(c-C₆H₁₁NH₂)(OH₂)]⁺ (2), and trans-[PtCl(NH₃)(quinoline)(OH₂)]⁺ (3). The reaction kinetics were studied at pH 6.0, 25 °C, and 1.0 mM ≤ I ≤ 500 mM. For both types of nucleic acid targets, compound 3 was found to react about 1 order of magnitude more rapidly than compounds 1 and 2. Further, all platinum compounds exhibited a more pronounced salt dependence for the interaction with r(CGCGUUGUUCGCG). Chemical and enzymatic cleavage studies revealed similar interaction patterns with r(CGCGUUGUUCGCG) after long exposure times to 1 and 2. A substantial decrease of cleavage intensity was found at residues G4 and G7, indicative of bifunctional adduct formation. Circular dichroism studies showed that platinum adduct formation leads to a structural change of the ribonucleic acid. Thermal denaturation studies revealed platination to cause a decrease of the RNA melting temperatures by 5–10 °C. Our observations therefore suggest that RNA is a kinetically competitive target to DNA. Furthermore, platination causes destabilization of RNA structural elements, which may lead to deleterious intracellular effects on biologically relevant RNA targets.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The use of calorimetry in the biophysical characterization of small molecule alkaloids binding to RNA structures

BackgroundRNA has now emerged as a potential target for therapeutic intervention. RNA targeted drug design requires detailed thermodynamic characterization that provides new insights into the interactions and this together with structural data, may be used in rational drug design. The use of calorimetry to characterize small molecule–RNA interactions has emerged as a reliable and sensitive tool after the recent advancements in biocalorimetry.Scope of the reviewThis review summarizes the recent advancements in thermodynamic characterization of small molecules, particularly some natural alkaloids binding to various RNA structures. Thermodynamic characterization provides information that can supplement structural data leading to more effective drug development protocols.Major conclusionsThis review provides a concise report on the use of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques in characterizing small molecules, mostly alkaloids–RNA interactions with particular reference to binding of tRNA, single stranded RNA, double stranded RNA, poly(A), triplex RNA.General significanceIt is now apparent that a combination of structural and thermodynamic data is essential for rational design of specific RNA targeted drugs. Recent advancements in biocalorimetry instrumentation have led to detailed understanding of the thermodynamics of small molecules binding to various RNA structures paving the path for the development of many new natural and synthetic molecules as specific binders to various RNA structures. RNA targeted drug design, that remained unexplored, will immensely benefit from the calorimetric studies leading to the development of effective drugs for many diseases. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

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.     

Fluorescence-based duplex-quadruplex competition test to screen for telomerase RNA quadruplex ligands

RNA and DNA guanine-rich sequences can adopt unusual structures called Guanine quadruplexes (G4). A quadruplex-prone RNA sequence is present at the 5'-end of the 451-nt-long RNA component of telomerase, hTERC. As this quadruplex may interfere with P1 helix formation, a key structural element for this RNA, we are seeking molecules that would alter this RNA duplex-quadruplex equilibrium. In this work, we present a fluorescence-based test designed to identify G4 ligands specific for the hTERC G-rich motif and that can prevent P1 helix formation. From an initial panel of 169 different molecules, 11 were found to be excellent P1 duplex inhibitors. Interestingly, some of the compounds not only exhibit a strong selectivity for quadruplexes over duplexes, but also demonstrated a preference for G4-RNA over all other quadruplexes. This test may easily be adapted to almost any quadruplex-forming sequence and converted into HTS format.     

A COMPARISON OF THE PROPERTIES AND THE SOLUTION STRUCTURE FOR RNA AND DNA QUADRUPLEXES WHICH CONTAIN TWO GGAGG SEQUENCES JOINED WITH A TETRANUCLEOTIDE LINKER

Abstract

We have determined solution structure of r(GGAGGUUUUGGAGG) (R14) by NMR; the RNA 14-mer forms an intra-strand parallel quadruplex with a G-tetrad and a hexad, in which a G-tetrad core is augmented by association of two A residues. The quadruplex further forms a dimer through stacking interaction between the hexads. In order to obtain insight into the difference between RNA and DNA quadruplexes, we synthesized the corresponding DNA 14-mer, d(GGAGGTTTTGGAGG) (D14), and examined its properties and structure by CD, gel electrophoresis, and NMR. K⁺ ions increased the thermal stability of both R14 and D14 structures. The binding affinity of K⁺ ions to R14 was much higher than that to D14. The CD and gel electrophoretic studies suggest that D14 forms a quadruplex entirely different from that of R14 in the presence of K⁺ ions; two molecules of D14 form a quadruplex with both antiparallel and parallel strand alignments and with diagonal loops at both ends of the stacked G-tetrads. The NMR study also gave results that are consistent with such structure: alternate glycosidic conformation, 5′G(syn)-G(anti)3′, and characteristic chemical shift data observed for many quadruplexes containing diagonal TTTT loops.     

Cleavage of RNA in Hybrid Duplexes by E. coli Ribonuclease H: III. Substrate Properties of RNA Duplex Complexes with Oligodeoxyribonucleotides Containing a Bleomycin A5 Residue

The effect of bleomycin A₅ residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E. coli RNase H. The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides. The RNA in the bleomycin-containing hybrid duplexes is cleaved by E. coli RNase H; however, the initial hydrolysis rate (v ₀) is 2.6–3.4-fold reduced for the continuous duplexes. In the case of tandem hybrid complexes, the effect of bleomycin on v ₀ was less pronounced. We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes. Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties.     

Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

Abstract

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg²⁺ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements.

Increased concentration of Mg²⁺ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na⁺ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg²⁺.     

Thermodynamic Features of Structural Motifs Formed by β-L-RNA

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2′-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by ¹H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.     

Synthesis of Modified RNA-Oligonucleotides for Structural Investigations

Abstract

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) and 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U- rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.