Poly(2-amino-8-methyladenylic acid). Competing structural and energetic effects of substituents

The ribopolynucleotide poly(2-amino-8-methyladenylic acid), (r2NH2(8)MeA)n, has been synthesized, and its physical and chemical properties have been examined. The study reveals competing effects on these properties of the 2-NH2 and 8-Me substituents. In marked contrast to the analogous (r8MeA)n, the new polymer readily interacts to form double helixes with complementary pyrimidine polynucleotides. Triple helixes are not formed. The 8-Me group is strongly destabilizing for helix formation (delta Tm approximately 65 degrees C), presumably by favoring a syn conformation, which blocks heteroduplex formation with ribohomopolymers. The 2-NH2 substituent stabilizes helixes in the ribo series by about 30 degrees C in Tm by forming a third interbase hydrogen bond. We suggest that the free energy from the 2-NH2 interaction drives the syn-anti equilibrium to the purine polymer to the anti form present in the double helix. CD spectra of the homopolymers (r2NH2A)n and (r2NH2(8)MeA)n are completely different, reflecting major differences of conformation. The double helixes formed by these polymers with (rT)n and (rBrU)n, on the other hand, have closely similar CD spectra, supporting our proposal of a major change in conformation of (2NH2(8)MeA)n on going from single strand to double helix.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

Structure of the deoxytetranucleotide d-pApTpApT and a sequence-dependent model for poly(dA-dT)

The x-ray structure of the hydrated ammonium salt of the deoxytetranucleotide d-pApTpApT was determined by Patterson and direct methods at a resolution of 1 Å. The crystal structure contains right-handed double-helical segments formed by complementary Watson-Crick-type hydrogen bonding between the adenine and thymine bases of neighboring molecules. The minihelix contains two base pairs. The chains are antiparallel. The A-T and T-A sequences have different phosphodiester conformations. The deoxyribose-pucker and the sugar–base orientation alternate along the chain depending on the nature of the base (3′-endo for purine and 2′-endo for pyrimidine). The extended structure is stabilized by base–base, base–sugar, and hydrogen-bond interactions. The minihelix of two base pairs provides starting coordinates for model-building studies of the dA-dT polymer. A B-DNA-type polymer structure is described, which has sequence-dependent alternations of both the deoxyribose pucker and the phosphate diester bridge conformation. Such sequence-dependent DNA structures, if present locally in regions such as operator sequences, could facilitate sequence-specific interactions. The crystal study also suggests possible geometrical parameters for the replication fork.     

Effects of monofunctional platinum binding on the thermal stability and conformation of a self-complementary 22-mer

We investigated the effect of various monofunctional platinum complexes on the thermal stability and conformation of a self-complementary 22-mer duplex oligonucleotide by means of CD and UV melting profiles. We studied several families of triamine complexes of the general formula PtA2AmCl where A2=(NH3)2 and ethylenediamine and where Am=N1-4-methyl-pyridine, N7-guanosine, and 9-ethyl-guanine. Platination by the N1-4-methyl-pyridine and 9-ethyl-guanine complexes led to a decrease in the Tm of the oligonucleotide by 2-11.5 degrees C while platination with the N7-guanosine complexes led to a rise in the melting temperature of the oligonucleotides by 4.5 degrees C. A similar inverse correlation between the two groups of platinum compounds was found in the CD spectra. In all cases, the cis isomer had a more pronounced effect on both the melting curve and the CD spectrum. The cis isomer was found to have a more destabilizing effect than its trans counterpart. This indicates that the cis geometry in fact forces a greater structural constraint on the backbone of the double helix. We have also found that the sugar of the guanosine has a significant influence on both the Tm and CD spectra; the sugar moiety contributes to the stability of the double helix, probably through the formation of hydrogen bonds.     

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.     

Raman and Raman optical activity (ROA) analysis of RNA structural motifs in Domain I of the EMCV IRES

Raman and Raman optical activity (ROA) spectra were collected for four RNA oligonucleotides based on the EMCV IRES Domain I to assess the contributions of helix, GNRA tetraloop, U·C mismatch base pair and pyrimidine-rich bulge structures to each. Both Raman and ROA spectra show overall similarities for all oligonucleotides, reflecting the presence of the same base paired helical regions and GNRA tetraloop in each. Specific bands are sensitive to the effect of the mismatch and asymmetric bulge on the structure of the RNA. Raman band changes are observed that reflect the structural contexts of adenine residues, disruption of A-form helical structure, and incorporation of pyrimidine bases in non-helical regions. The ROA spectra are also sensitive to conformational mobility of ribose sugars, and verify a decrease in A-type helix content upon introduction of the pyrimidine-rich bulge. Several Raman and ROA bands also clearly show cooperative effects between the mismatch and pyrimidine-rich bulge motifs on the structure of the RNA. The complementary nature of Raman and ROA spectra provides detailed and highly sensitive information about the local environments of bases, and secondary and tertiary structures, and has the potential to yield spectral signatures for a wide range of RNA structural motifs.     

Synthesis and triplex binding properties of oligonucleotides containing a novel nucleobase

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

Circular dichroism of flow-oriented nucleic acids. II. Comparison between observed and calculated spectra.

The ultraviolet circular dichroism (CD) of oriented DNA and RNA molecules is calculated by an extension of Johnson and Tinoco's theory [(1969) Biopolymers 7 , 727–749] for unoriented molecules. The calculations are carried out for molecular models of A-DNA, B-DNA, planar B-DNA, C-DNA, and RNA-11-α. The calculated curves are compared to measured spectra [(1975) J. Mol. Biol. 92 , 449–466] Chung and Holzwarth, for oriented solutions of DNA in buffer, DNA in 6 M LiCl or in ethylene glycol, and double-stranded viral RNA. The calculation, which considers only base–base interactions, predicts that the CD of B-DNA, measured with light propagating parallel to the helix axis, should be large and semiconservative, whereas the CD for light propagating perpendicular to the helix axis should be nonconservative. These predictions agree qualitatively with the experimental observations for DNA in buffer; agreement improves if one assumes the bases to be exactly perpendicular to the helix axis. For the other geometries, agreement is less satisfactory, but qualitative agreement with experiment is obtained and the signs of the specific CD spectra are in accord with observations.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions

Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).     

Hydrogen-bonded complexes of the ribodinucleoside monophosphates in aqueous solution. Proton magnetic resonance studies.

A proton magnetic resonance study of the chemical shifts of a series of ribodinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range. The self-complementary dinucleoside monophosphates CpG and GpC and the complementary mixture GpU + ApC form intermolecular hydrogen-bonded complexes at low temperatures. The amino proton chemical shifts in the CpG and GpC spectra are consistent with the formation of a miniature double helical dimer in neutral aqueous solution at low temperatures (approximately 2 degrees C). The complementary mixture of dinucleosides GpU + ApC formed much less stable complexes than either GpC or CpG, while UpA did not show any indication of the formation of intermolecular hydrogen-bonded complexes. This result is consistent with the well-known observation that the stability of a double helix is proportional to the percent of G-C base pairs present.     

Structure and dynamics of a DNA-based model system for the study of electron spin-spin interactions

We report on the structure and dynamics of a model system for measuring long-range distances in biological macromolecules by saturation-recovery EPR. Four DNA duplexes that incorporate a paramagnetic dysprosium ion (Dy(III)) and a nitroxide spin-label were examined by electron paramagnetic resonance (EPR), circular dichroism (CD), and ultra-violet absorbance (UV) spectroscopy. Dy(III) is chelated by the modified base deoxythymidine-EDTA, (dT-EDTA). Electron spin-spin interactions between the Dy(III) ion and the nitroxide radical are observed at distances as great as ∼5.3 nm. A slight change in the conformation of those nucleotides lying between the EDTA(Dy(III)) complex and the nitroxide spin-label results in a “stiffening” of the DNA helix on the EPR time scale. Changes in conformation and helix dynamics are due to the binding of the EDTA(Dy(III)) complex to the phosphodiester backbone of the complementary strand. Molecular mechanics calculations indicate that binding occurs in the 5′ direction on the complementary strand, at a position 3 or 4 phosphates distant from the dT-EDTA(Dy(III)) * dA base pair.     

Synthesis and Triplex Binding Properties of Oligonucleotides Containing a Novel Nucleobase

Abstract

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

Poly(8-bromodeoxyadenylic acid): properties of the polymer and contrast with the ribopolynucleotide analogue

Introduction of the bulky 8-bromo substituent into adenine residues of polynucleotides has strikingly different consequences in the deoxy- and ribopolynucleotide series. Poly(r8BrA) was found in earlier studies to form a very stable double-helical self-structure but not to undergo interaction with potentially complementary polynucleotides. We find that poly(d8BrA), in contrast, does not form an ordered self-structure in 0.1 M Na+ but appears to exist as an electrostatically expanded rigid rod with unusual circular dichroism (CD) properties at very low ionic strength. The deoxy polymer, moreover, readily forms double helices with either deoxy or ribo pyrimidine polynucleotides, studied by UV, CD, and IR spectroscopy. These complexes are destabilized, relative to those formed by poly(dA), possibly because energy is needed to convert the purine residues from a more stable syn to an anti conformation, required for heteroduplex formation. The CD spectrum of (d8BrA)n X (dT)n is similar to that of B DNA. The deoxy-ribo hybrids (d8BrA)n X (rU)n and (d8BrA)n X (rBrU)n have CD spectra resembling those of A DNA or RNA. Unlike other deoxy-deoxy pairs (d8BrA)n X (dBrU)n, however, has a CD spectrum resembling RNA and other helices having the A form.     

肽核酸在分子生物学技术中的应用

肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同。PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构。与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点。简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望。     

Sequence-dependent structural variations in two right-handed alternating pyrimidine-purine DNA oligomers in solution determined by nuclear Overhauser enhancement measurements

A 500 MHz 1H-n.m.r. study on two right-handed self-complementary double-stranded alternating pyrimidine-purine oligodeoxyribonucleotides, 5'dCGTACG and 5'dACGCGCGT, is presented. Using the proton-proton nuclear Overhauser effect, proton resonances are assigned by a sequential method and a large number of interproton distances, both intra- and internucleotide, are determined (113 for 5'dCGTACG and 79 for 5'dACGCGCGT). The general procedure required to solve the three-dimensional solution structures of oligonucleotides from such distance data is outlined and applied to these two oligonucleotides. In the case of both oligonucleotides the overall solution structure is that of B DNA, namely a right-handed helix with a helical rise of approximately 3.3 A, 10 bp per turn and the base pairs approximately perpendicular to the helix axis. In the case of 5'dCGTACG, subtle local structural variations associated with the pyrimidine and purine nucleotides are superimposed on the overall structure but the mononucleotide repeating unit is preserved. In contrast, 5'dACGCGCGT has a clear alternating structure with a dinucleotide repeat, alternation occurring in the local helical twist and the glycosidic bond, sugar pucker and phosphodiester backbone conformations.     

Far-ultraviolet optical activity of saccharide derivatives. II. Dextran,amylose, and mycodextran acetes; dextran and amylose xanthates

The ultraviolet ORD and CD spectra of amylose, dextran, and mycodextran acetates and some of thier oligomers were recorded in trifluoroethanol solution in the 300–185nm wavelength range. Similarly, the spectra of amylose and dextran xanthates in water solution were obtained in the 400–200 nm range. In the amylose acetate series, the monomer and dimer both show a normal acetyl n → π* transition in CD, while the trimer and the polymer both exhibit an additional, shorter wavelength peak. The latter is presumed to arise from a helical conformation of the amylose chain. This interpretation is substantiated by a helix–coil type transition of the CD spectra of amylose triacetate at elevated temperatures and a reversion of the anomalous CD to the normal CD upon partial deacetylation. By contrast, neither dextran acetates nor mycodextran acetate exhibit any conformational effects. The CD of dextran acetates is quite sensitive to β-1,6 and branch linkages. The ORD and CD of amylose xanthate are complex, suggesting the presence of organized structure in solution. The dextran xanthate shows only a simple ORD spectrum and no observable CD.     

DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization

The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7°C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5′-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 ± 0.17 Å. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.     

Structure-wise discrimination of cytosine,thymine, and uracil by proteins in terms of their nonbonded interactions

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein–ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue–ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.