Backbone Linkages Research Articles

Structure of a stereoregular phosphorothioate DNA/RNA duplex.

In this work, we present the first NMR solution structure of a DNA/RNA hybrid containing stereoregular Rp-phosphorothioate modifications of all DNA backbone linkages. The complex of the enzymatically synthesized phosphorothioate DNA octamer (all-Rp)-d(GCGTCAGG) and its complementary RNA r(CCUGACGC) was found to adopt an overall conformation within the A-form family. Most helical parameters and the sugar puckers of the DNA strand assume values intermediate between A- and B-form. The close structural similarity with the unmodified DNA/RNA hybrid of the same sequence may explain why both the natural and the sulfur-substituted complex can be recognized and digested by ribonuclease H.

Optical anisotropy of comb-like macromolecules of lateral and semilateral structure

Maxwell effect and Kerr effect techniques were used to study the intramoleculare axial ordering of anisotropic side groups of an isolated macromolecule in solution. Three comb-like polymers with a methacrylic main chain were investigated. The side chains contain aliphatic spacers separating the mesogenic fragments R from the backbone. R contains two or three phenylene rings with ester linking groups and cyano end groups. Fragments R are attached to the side chains, by the end or middle-point, through the para- or ortho-phenylene ring. The main-chain conformation in solution was identified to assess the hydrodynamic data using the Gaussian coil approximation. The optical anisotropy of the macromolecule segment was determined and related to the monomer unit structure. The specific Kerr constant obtained in diluted benzene solution of polymer was compared with those of low molecular weight analogues of the polymer mesogenic groups. The mobility and intramolecular ordering of the mesogenic groups in the isolated macromolecules are discussed. These properties are compared with those of comb-like polymers with another type of attachment of mesogenic groups, and with another (siloxane) chemical structure of the backbone chain. The differences in restrictions of side-chain mobility due to the backbone linkage in different types of comb-like polymers are also discussed.

Optical anisotropy of comb-like macromolecules of lateral and semilateral structure

Maxwell effect and Kerr effect techniques were used to study the intramoleculare axial ordering of anisotropic side groups of an isolated macromolecule in solution. Three comb-like polymers with a methacrylic main chain were investigated. The side chains contain aliphatic spacers separating the mesogenic fragments R from the backbone. R contains two or three phenylene rings with ester linking groups and cyano end groups. Fragments R are attached to the side chains, by the end or middle-point, through the para- or ortho-phenylene ring. The main-chain conformation in solution was identified to assess the hydrodynamic data using the Gaussian coil approximation. The optical anisotropy of the macromolecule segment was determined and related to the monomer unit structure. The specific Kerr constant obtained in diluted benzene solution of polymer was compared with those of low molecular weight analogues of the polymer mesogenic groups. The mobility and intramolecular ordering of the mesogenic groups in the isolated macromolecules are discussed. These properties are compared with those of comb-like polymers with another type of attachment of mesogenic groups, and with another (siloxane) chemical structure of the backbone chain. The differences in restrictions of side-chain mobility due to the backbone linkage in different types of comb-like polymers are also discussed.

Optical anisotropy of comb-like macromolecules of lateral and semilateral structure

Maxwell effect and Kerr effect techniques were used to study the intramoleculare axial ordering of anisotropic side groups of an isolated macromolecule in solution. Three comb-like polymers with a methacrylic main chain were investigated. The side chains contain aliphatic spacers separating the mesogenic fragments R from the backbone. R contains two or three phenylene rings with ester linking groups and cyano end groups. Fragments R are attached to the side chains, by the end or middle-point, through the para- or ortho-phenylene ring. The main-chain conformation in solution was identified to assess the hydrodynamic data using the Gaussian coil approximation. The optical anisotropy of the macromolecule segment was determined and related to the monomer unit structure. The specific Kerr constant obtained in diluted benzene solution of polymer was compared with those of low molecular weight analogues of the polymer mesogenic groups. The mobility and intramolecular ordering of the mesogenic groups in the isolated macromolecules are discussed. These properties are compared with those of comb-like polymers with another type of attachment of mesogenic groups, and with another (siloxane) chemical structure of the backbone chain. The differences in restrictions of side-chain mobility due to the backbone linkage in different types of comb-like polymers are also discussed.

Peptide nucleic acids: Expanding the scope of nucleic acid recognition

Peptide nucleic acids (PNAs) are DNA analogs containing neutral amide backbone linkages. PNAs are stable to degradation by enzymes and hybridize to complementary sequences with higher affinity than analogous DNA oligomers. PNA synthesis employs protocols derived from solid-phase peptide synthesis, making the methodology straightforward and flexible. PNAs are being incorporated into an expanding set of applications, including genome mapping, the identification of mutations and measurement of telomere length. The growth in the popularity of PNAs as a tool for nucleic acid recognition should accelerate as the properties of PNAs become more familiar.

Conformational studies of antisense DNA by PFG NMR.

Pulsed field gradient diffusion constant measurements were used to resolve the ambiguity in determining the conformational states of single-stranded DNA dodecanucleotides (d1s, d4s and d5s). For d1s and d5s, because of the spectral symmetry conventional NMR analyses cannot differentiate whether they are hairpins or homo-duplexes. However, the diffusion constants of these sequences at 300 K are 1.4 times greater than those of the comparison complementary duplexes. This result agrees well with what is expected for Dhairpin/Dduplex based on classic liquid-phase translational diffusion models and the Einstein-Stokes equation, confirming that d1s and d5s form hairpins. d4s did not show a structured spectral pattern, but its diffusion constant measurement suggests that this sequence may not be a random coil. The DNA sequences studied contain chemically modified backbone linkages and are potential antisense agents for gene regulation. The knowledge of their diffusion constants, in combination with conventional NMR analysis and other biophysical spectroscopic measurements, provides new insights into the relationships of chemical structure and conformational preference of antisense oligonucleotides and their analogs.

PHOSPHONOACETATE DERIVATIVES OF OLIGODEOXYRIBONUCLEOTIDES

Abstract A deoxyribodinucleotide phosphonoacetate derivative has been prepared, separated into individual diastereomers, and incorporated into oligodeoxyribonucleotides possessing alternating phosphodiester and phosphonoacetate backbone linkages. The hybridization properties and enzymatic stabilities of these oligonucleotides have been studied.

Concentration and Extinction Coefficient Determination for Oligonucleotides and Analogs Using a General Phosphate Analysis

A general phosphate analysis (GPA) is developed which assays the concentration of nucleic acid oligomers and their analogs based on stoichiometric phosphorus in the sequence. The method involves complete digestion of the oligomer sample to orthophosphate using acid at high temperature and subsequent colorimetric analysis by phosphomolybdate complex formation. GPA is applied to oligomers having phosphodiester, methylphosphonate, and phosphorothioate backbone linkages. Given the absorption spectra of oligomers having these backbones, extinction coefficients are obtained and compared to other quantitative and predictive methods. In addition to sequences having the usual nucleoside residues found in naturally occurring nucleic acids, oligomers having base analog residues can be readily quantified by GPA.

Nuclease Resistance and Antisense Activity of Modified Oligonucleotides Targeted to Ha-ras

We have previously described structure-activity studies on a 17-mer uniform phosphorothioate antisense sequence targeted to human Ha-ras. In an effort to further improve the pharmacological properties of antisense oligonucleotides, structure-activity studies on this 17-mer sequence were expanded to examine both the effects of replacing phosphorothioate backbone linkages with phosphodiester linkages and the effects of incorporating various 2'-sugar modifications into phosphorothioate and phosphodiester oligonucleotides on oligonucleotide stability against nucleases in vitro and on antisense activity in cells. Replacement of three or more phosphorothioate linkages with phosphodiester linkages greatly compromised both nuclease resistance and antisense activity, and these effects correlated directly with the number of phosphodiester linkages incorporated into the oligonucleotide. However, substantial nuclease resistance, sufficient for obtaining potent antisense effects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2'-alkoxy sugar modifications. Nuclease stability and antisense activity imparted by these sugar modifications in phosphodiester backbones correlated with the size of the 2'-alkoxy substituent (pentoxy > propoxy > methoxy > deoxy). Furthermore, antisense activity mediated by oligonucleotides that exhibit partial resistance to nucleolytic degradation was dependent on both oligonucleotide concentration and the duration of oligonucleotide treatment.

Structure of a DNA duplex that contains alpha-anomeric nucleotides and 3'-3' and 5'-5' phosphodiester linkages: coexistence of parallel and antiparallel DNA.

We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.

Synthesis of a New Hydroxyamino Linked Thymidine Dimer via a Radical C-C Bond Formation

Abstract An efficient synthesis of a thymidine nucleoside dimer [T-3′-β-O-N(CH3)-CH2-5′-T] has been accomplished via an intermolecular radical coupling reaction. The novel dimer contains an achiral and neutral backbone linkage which may have potential application in constructing backbone modified antisense oligonucleosides.

Synthesis, Biophysical and Biological Evaluations of Novel Antisense Oligonucleosides Containing Dephosphono-Internucleosidic Linkages

Abstract Efficient large-scale syntheses of methylene(methylimino) (MMI) linked mixed base nucleosidic dimers have been accomplished. These dimers were successfully incorporated into deoxyoligonucleotides by automated solid-support synthesis. The hybridization properties, nuclease stability, RNase H mediated cleavage and in vitro biological activity of novel chimeric oligomers have been studied. The biophysical and biological evaluation of these chimeric oligomers containing MMI linkages suggests that MMI is a promising chemical modification of the backbone linkage for the construction of antisense molecules useful as therapeutics.

Crystal structure and NMR conformation of a cyclic pseudotetrapeptide containing urethane backbone linkages.

Urethane bonds, derived from the hydroxyl group of the tyrosine side chain, have been investigated as a new type of amide bond mimetic in the design of pseudopeptides. The structure of a representative cyclic pseudotetrapeptide that consists of an -Ala-Tyr(urethane)Ala-Tyr(urethane) sequence fused into a rigid ring has been studied in the solid state by x-ray crystallography and in solution by two-dimensional nmr techniques. The cyclic pseudotetrapeptide has an oblong shape. The backbone urethane bonds assume a trans-trans conformation. The carbonyl groups in the ring have an alternating pattern of down, up, down, up with respect to the average ring plane. Solution nmr studies give observed nuclear Overhauser effects and coupling constants largely in agreement with the crystal structure. However, in solution the observed structure is likely to be conformationally averaged, and in the averaged structure, the urethane bond is perpendicular to the plane of the aromatic ring of the tyrosine, while in the crystal it is close to this plane. These differences may be explained by intermolecular hydrogen-bonding interactions. Four aspects of the conformation of the cyclic pseudotetrapeptide were investigated in detail: the tyrosine residue with the attached side-chain urethane bond (the tyrosine-urethane unit), the conformation of the two urethane backbone linkages, the conformation of the two conventional peptide bonds within this unusual ring structure, and the tight turns within the cyclic pseudotetrapeptide. The conformation of the tight turns present in the cyclic pseudotetrapeptide is very similar to that of a beta-bend of type II. Intermolecular hydrogen bonding, joining adjacent layers of the cyclic pseudotetrapeptide in the solid state, resemble a parallel beta-pleated sheet. The presence of these structural motifs in the cyclic pseudotetrapeptide indicates that the tyrosine urethane unit may find applications in peptide and protein engineering.

Metabolic processing of cyclobutyl pyrimidine dimers and (6-4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.

A new nuclease digestion assay was developed to elucidate the human excision-repair system operating on cyclobutyl pyrimidine dimers and (6-4) photoproducts. We analyzed lesions that accumulated in excised oligonucleotide fragments during incubation of UV-treated cultured fibroblasts. (6-4) photoproducts were removed intact, whereas excised cyclobutyl dimers often contained ruptured interpyrimidine phosphodiester bonds, raising the possibility that the intradimer backbone-cleavage reaction may help promote the bypass of unexcised dimers by the DNA replication or RNA transcription machinery. Cell strains representing eight different inherited forms of the cancer-prone skin disease xeroderma pigmentosum (XP) were generally found to exhibit characteristic abilities to excise the two classes of photolesions, ranging from total deficiency in groups A and G to normal proficiency in the variant. The capacity of any given XP group to act on one class of photoproducts in no way predicted its ability to act on the other. Finally, in those XP strains displaying significant levels of dimer removal, the ratio of intact-versus-modified dimers was normal, implying that rupture of the intradimer backbone linkage occurs independently of subsequent excision-repair reactions. Our data indicate that cyclobutyl dimers and (6-4) photoproducts are processed by distinct nucleotide-excision-repair pathways in human cells.

Unusual conformation of a 3?-thioformacetal linkage in a DNA duplex

The DNA.DNA duplex d(CGCGTTSCH2OTTGCGC).d(GCGCAAAACGCG) (designated duplex III) containing a 3'-thioformacetal (3'-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH2O) linkage at the central T--T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3'-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the T6(3'-SCH2O)T7 modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3'-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3'-TFMA-modified T6 sugar adopts an O4'-endo form (an intermediate structure between the usual C2'-endo and C3'-endo forms). This change is accompanied by a change in the epsilon (C4'-C3'-S3'-CH2) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6-T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3'-TFMA linkages is merely in the substitution of the T6(O3') in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.

Purification and properties of a xanthan depolymerase from a heat-stable salt-tolerant bacterial consortium

A bacterial consortium (NRRL B-14401) resulting from soil enrichment growth on xanthan gum produces enzymes that can degrade xanthan gum in salt-containing solutions at temperatures up to 65°C. One component that cleaves the backbone linkages of both xanthan gum and carboxymethyl cellulose is called xanthan depolymerase. Two such depolymerase activities were isolated by high performance anion exchange chromatography, and their molecular weights determined by size exclusion chromatography to be 170 000 and 100 000 Da. The 170-kDa protein was purified and its properties studied. Sodium chloride and potassium chloride enhanced the hydrolysis of carboxymethyl cellulose, but decreased the rate of degradation of xanthan gum. The purified enzyme, which was optimally active at pH 6, was less stable to extremes of temperature than crude mixtures of cell-free culture broth; stabilized by i substrate it was active for more than 6 h at 50°C.

Solution structure of [d(GCGTATACGC)] 2

The solution structure of the alternating pyrimidine-purine DNA duplex [d(GCGTATACGC)] 2 has been determined using two-dimensional nuclear magnetic resonance techniques and distance geometry methods. Backbone distance constraints derived from experimental nuclear Overhauser enhancement and J-coupling torsion angle constraints were required to adequately define the conformation of the inter-residue backbone linkages and to avoid underwinding of the duplex. The distance geometry structures were further refined by back-calculation of the two-dimensional nuclear Overhauser enhancement spectra to correct spin-diffusion distance errors. Fifteen final structures for [d(GCGTATACGC)] 2 were generated from the refined experimental distance bounds. These structures all exhibit fully wound B-form geometry with small penalty values (< 1.5 Å) against the distance bounds and small pair-wise root-mean-square deviation values (typically 0.6 Å to 1.5 Å). The final structures exhibit positive base-pair inclination with respect to the helix axis, a marked alternation in rise and twist, and are shorter and wider than classical fiber B-form DNA. The purines were found to adopt a sugar pucker close to the C-2′- endo conformation while pyrimidine sugars exhibited significantly lower pseudorotation phase angles in the C-1′- exo to C-2′- endo range. The minor groove crossstrand steric clashes at pyrimidine-purine steps that would exist in pure B-DNA are attenuated by an increased rise at these steps (and an increased roll angle at TpA steps). Concomitantly the backbone torsion angles of the pyrimidine moieties have larger γ values, larger ε values, and smaller ζ values than the purines. The structures generated by distance geometry methods were also compared with those obtained from restrained molecular dynamics with empirical force-field potentials. The results indicate that the nuclear magnetic resonance/distance geometry approach alone is capable of elucidating most of the salient structural features of double-stranded helical nucleic acids in solution without resorting to empirical energy potentials and without using any structural assumptions from crystallographic data.

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.

Dynamics of controlled release from bioerodible matrices

Abstract A mathematical model for the analysis of the basic physicochemical determinants that yields experimentally verifiable predictions of controlled release of bioactive agents from eroding polymeric matrices is presented. This model is applied to the erosion characteristics of acid catalyzed erosion of poly (ortho ester)s since there is considerable information in the literature on the detailed physical and chemical performance of this system. The analysis shows that the dynamic changes in polymer matrix properties (namely, simultaneous reaction-diffusion-transport of matrix constituents, moving diffusion and water fronts, water-polymer partition coefficients, solubility of water and diffusivity of matrix components as a function of the extent of acid catalyzed matrix hydrolysis) play a significant role in regulating the release kinetics of bioactive agents. The analysis of poly (ortho ester) erosion as a test system predicts experimentally verifiable measurable quantities: release characteristics of the incorporated bioactive agent, water penetration into the matrix, catalysis by the anhydride and catalytic degradation of the polymer matrix. Further, an estimate of the concentration of unbroken polymer backbone linkages and hence the molecular weight of the polymer disc with time is obtained using random scission kinetics. The Thiele modulus is a good indicator of surface versus bulk erosion and has been successfully applied to characterize the erosion characteristics of the poly (ortho ester) system. Finally, an analysis for the basic design, interpretation, and prediction of overall release modes (bulk versus surface erosion) from devices that rely on simultaneous reaction and diffusion controlled erosion phenomenon is presented.

Hybridization Properties of a DeoxyoIigonucleotide Containing Four Formacetal Linkages

Abstract The deoxyoiigonucleotide “TCTCTCTCTCT” bearing four formacetal linkages has been synthesized. The hybridization properties of this compound with complementary RNA and single stranded DNA has been compared to control sequences bearing phosphodiester, phosphoramidate and methylphosphonate linkages. The formacetal oligomer hybridizes to RNA similarly to a diester and significantly better than the other neutral linkages. Recently we reported the synthesis of a deoxyoligonucleotide bearing two formacetal linkages at the three prime end (1). This oligomer was characterized for binding to a complementary RNA sequence. We now report the synthesis of the sequence 5′ TCTC∗TC∗TC∗TCTTT where C is 5 methyl deoxycytidine and ∗ is the formacetal linkage. Controls were prepared in which the modified linkage position ∗ was the normal phosphodiester, methylphosphonate and methoxyethyl phosphoramidate. These four sequences which differ only in the backbone linkage at the starred positions were compared for their ab...