Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.     

Thermodynamic analysis of duplex formation of the heterochiral DNA with L-deoxyadenosine.

An L-DNA, the mirror-image isomer of natural DNA, has extraordinary nuclease resistance, and thus the molecules should be promising reagents for many applications, such as antisense technology. However, little is known about the structural and thermodynamic properties of DNAs with this modified nucleotide. In this study, we prepared the L-nucleotide (L-dA) and introduced it into oligodeoxyribonucleotides to assess the ability of the L-nucleotide as a functional molecule for many applications based on the DNA hybridization. Two decamers with an L-dA at the center were synthesized and duplexes with the complementary DNA strand were applied to structural and thermodynamic analyses. The structural study by CD spectra showed that the structures of both modified "L/D-D" duplexes were the typical B-form. This result suggests that the global structure of DNA was not collapsed by the introduction of an L-DNA. Thermodynamic parameters (deltaH degrees, deltaS degrees, and deltaG degrees 37) of the duplex formation, determined by UV melting experiments, indicated that the both duplexes were destabilized at about 2.5 to 3.0 kcal mol(-1) by the introduced L-dA, mainly due to an unfavorable enthalpic effect. In conjunction with information by other researchers, these results suggest that the L-DNA affect on the duplex structure and the stability vary locally; thus, the thermodynamic stability of modified L/D-D duplexes should be predictable by the nearest-neighbor thermodynamic parameters.     

Determination of affinity constants of locked nucleic acid (LNA) and DNA duplex formation using label free sensor technology

Locked nucleic acid (LNA) is a nucleic acid analogue containing 2'-O,4'-C-methylene-beta-D-ribofuranosyl nucleotides, which have a bicyclic furanose unit locked in a RNA mimicking sugar conformation. Oligonucleotides containing LNA monomers show an enhanced thermal stability and robustness against nuclease mediated cleavage. Therefore special tailored LNA is a versatile tool for gene array analysis and single nucleotide polymorphism (SNP) analysis. The higher melting temperatures result from a higher affinity between the LNA and its complementary base. This was verified by the determination of the affinity constants of the duplex formation of 3 oligonucleotides: DNA, L-DNA, in which all thymidines are substituted by LNA, and a fully modified LNA, to their complementary DNA strand. Affinity constants were calculated to be 1.5 x 10(9), 4.0 x 10(9) and >10(12) L mol(-1). This was done using the label free and time resolved sensing technology reflectometric interference spectroscopy (RIfS), in an assay format similar to a titration called binding inhibition assay.     

Photoreactivity of UV-b damage in bacteriophage phi X174 DNA

Abstract— The fraction of biological damage in isolated single-strand and double-strand forms of bac-teriophage DNA resulting from pyrimidine dimers following exposure to germicidal UV (254 nm) and UV-B (280-320. nm) radiation has been compared. Radiation from a Westinghouse FS-40 sunlamp filtered through a cellulose acetate sheet was used as the UV-B radiation source. Biological damage from pyrimidine dimers was determined by measuring the survival of the viral DNA with and without photoreactivation, an enzymatic process specific for repair of pyrimidine dimers. The same fraction of biological damage in the single strand and double–strand forms of φX174 DNA is repairable by photo-reactivation following exposures to germicidal UV and UV-B radiation.     

铂类抗肿瘤药物ZD0473与双链及单链DNA作用差异的理论研究

用分子力学与量子化学相结合的MM/uff//HF/lanl2dz方法及单独的密度泛函方法探讨了新型铂类抗肿瘤药物(ZD0473)与碱基序列相同的双链及单链DNA片段几何结构、相互作用及电子结构方面的异同.结果表明,虽然药物分子都能与单、双链DNA形成稳定的复合物,但双链复合物与DNA形成的配位键比单链复合物稍强,氨中的氢与O6G₈间形成的氢键更强,甲基吡啶所造成的位阻效应更高,使得肿瘤细胞对药物的抗药性进一步降低.     

Helical self-organization and hierarchical self-assembly of an oligoheterocyclic pyridine-pyridazine strand into extended supramolecular fibers

The synthesis and characterization of an alternating pyridine-pyridazine strand comprising thirteen heterocycles are described. Spontaneous folding into a helical secondary structure is based on a general molecular self-organization process enforced by the conformational information encoded within the primary structure of the molecular strand itself. Conformational control based on heterocyclic "helicity codons" illustrates a strategy for designing folding properties into synthetic oligomers (foldamers). Strong intermolecular interactions of the highly ordered lock-washer subunits of compound 3 results in hierarchical supramolecular self-assembly into protofibrils and fibrils. Compound 3 also forms mechanically stable two-dimensional Langmuir-Blodgett and cast thin films.     

Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE

A new method has been developed allowing the identification and relative quantification of different forms of mRNA after RNA editing. This method was applied to the serotonin 2c receptor mRNA that potentially exhibits 32 different forms after adenosine to inosine editing at five different sites located in a row of 13 nucleotides. CE was used to characterize fluorescently labeled ssDNA molecules on the basis of their conformational polymorphism. The relative amount of these 32 mRNA forms has been estimated by measuring the fluorescence intensity of each individual DNA strand. Accuracy of quantification was established by diluting one form into another or into a mixture of cDNA, showing linear and precise proportion of each form (0.06 相似文献   

DNA zipper-based tweezers

Here we report the design and development of DNA zippers and tweezers. Essentially a zipper system consists of a normal strand (N), a weak strand (W), and an opening strand (O). N strand is made up of normal DNA bases, while W is engineered to have inosine substituting for guanine. By altering the number and order of inosine, W is engineered to provide less than natural bonding affinities to N in forming the [N:W] helix. When O is introduced (a natural complement of N), it competitively displaces W from [N:W] and forms [N:O]. This principle is incorporated in the development of a molecular device that can perform the functions of tweezers (sense, hold, and release). Tweezers were constructed by holding N and W together using a hinge at one end. Thus, when the tweezers open, N and W remain in the same vicinity. This allows the tweezers to cycle among open and close positions by their opening and closing strands. Control over their opening and closing kinetics is demonstrated. In contrast to the previously reported DNA tweezers, the zipper mechanism makes it possible to operate them with opening strands that do not contain single-stranded DNA overhangs. Our approach yields a robust, compact, and regenerative tweezer system that could potentially be integrated into complex nanomachines.     

Sequence specificity of hydrogen-bonded molecular duplexes

Hydrogen-bonded molecular duplexes, 1.3 and 1.4, each of which contains a mismatched binding site (acceptor-to-acceptor in 1.3, and donor-to-donor in 1.4), were designed and synthesized based on duplex 1.2. One- and two-dimensional NMR studies demonstrated that, despite their single mismatched binding sites, the backbones of duplexes 1.3 and 1.4 still stayed in register through the formation of the remaining five H-bonds. The backbones of 1.3 and 1.4 adjusted to the presence of the mismatched binding sites by slightly twisting around these sites, which alleviate any head-on repulsive interactions between two H-bond donors (amide O) or between two acceptors (amide H). After 1 equiv of single strand 2, which forms a perfectly matched duplex 1.2 with single strand 1, was added into the solution of either 1.3 or 1.4, only 1.2 and single strand 3 or 4, were detected. Isothermal titration calorimetry (ITC, in chloroform containing 5% DMSO) indicated that duplexes 1.3 and 1.4 were significantly (>40 times) less stable than the corresponding perfectly hydrogen-bonded duplex 1.2. These NMR and ITC results indicate that the pairing of two complementary single strands is not affected by another very similar single strand that contains only one wrong H-bond donor or acceptor, which demonstrates that the self-assembly of this class of H-bonded duplexes is a highly sequence-specific process. The role of these H-bonded duplexes as predictable and programmable molecular recognition units for directing intermolecular interactions has thus been established.     

Protein knots and fold complexity: some new twists

The current knowledge on topological knots in protein structure is reviewed, considering in turn, knots with three, four and five strand crossings. The latter is the most recent to be identified and has two distinct topological forms. The knot observed in the protein structure is the form that requires the least number of strand crossings to become un-knotted. The position of the chain termini must also correspond to a position that allows (un) knotting in one move. This is postulated as a general property of protein knots and other more complex knots with this property are proposed as the next most likely knots that might be found in a protein. It is also noted that the "Jelly-roll" fold found in some all-beta proteins would provide likely candidates. Alternative measures of knottedness and entanglement are reviewed, including the occurrence of slip-knots. These measures are related to the complexity of the protein fold and may provide useful filters for selecting predicted model structures.     

Kinetic and thermodynamic control of g-quadruplex folding

A matter of speed: When allowed to fold in a K(+) /poly(ethylene glycol) solution, the guanine?(G)-rich strand of vertebrate telomere DNA forms a parallel/antiparallel G-quadruplex, which is a (3+1) hybrid, within microseconds before slowly transforming into the parallel one within hours. Thus, the conformation that a G-quadruplex initially adopts under physiological conditions may not be the one it adopts at the equilibrium state.     

(3 + 1) Assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex

We present an NMR study on the structure of a DNA fragment of the human telomere containing three guanine-tracts, d(GGGTTAGGGTTAGGGT). This sequence forms in Na(+) solution a unique asymmetric dimeric quadruplex, in which the G-tetrad core involves all three G-tracts of one strand and only the last 3'-end G-tract of the other strand. We show that a three-repeat human telomeric sequence can also associate with a single-repeat human telomeric sequence into a structure with the same topology that we name (3 + 1) quadruplex assembly. In this G-quadruplex assembly, there are one syn.syn.syn.anti and two anti.anti.anti.syn G-tetrads, two edgewise loops, three G-tracts oriented in one direction and the fourth oriented in the opposite direction. We discuss the possible implications of the new folding topology for understanding the structure of telomeric DNA, including t-loop formation, and for targeting G-quadruplexes in the telomeres.     

EFFICIENCY OF REPAIR OF PYRIMIDINE DIMERS AND PSORALEN MONOADDUCTS IN NORMAL AND XERODERMA PIGMENTOSUM HUMAN CELLS

Abstract —Repair of DNA damage produced by ultraviolet light or 5-methylisopsoralen in normal and xeroderma pigmentosum human cells involves many similar steps. Aphidicolin and cytosine arabinoside block repair of both kinds of damage with similar efficiency, indicating that DNA polymerase a has a major role in repair for these lesions. In xeroderma pigmentosum cells of various complementation groups, the relative efficiency of excision repair for both ultraviolet- and 5-methylisopsoralen-induced damage was group A < C < D, indicating a close resemblance between both kinds of lesions in relation to the repair deficiencies in these groups. At high doses, the maximum rate of repair of damage by ultraviolet light was about twice that for methylisopsoralen damage, possibly because ultraviolet-induced damage forms a substrate that is more readily recognized and excised than that of the psoralen adducts. Differences in the structural distortions to DNA caused by these kinds of damage could be detected using single strand specific nucleases which excised dimers but not 5-MIP adducts from double strand DNA.     

Unexpectedly stable artificial duplex from flexible acyclic threoninol

A new foldamer, acyclic threoninol nucleic acid (aTNA), has been synthesized by tethering each of the genetic nucleobases A, G, C, and T to d-threoninol molecules, which were then incorporated as building blocks into a scaffold bearing phosphodiester linkages. We found that with its fully complementary strand in an antiparallel fashion, the aTNA oligomer forms an exceptionally stable duplex that is far more stable than corresponding DNA or RNA duplexes, even though single-stranded aTNA is rather flexible and thus does not take a preorganized structure.     

Electrochemical determination of triple helices: electrocatalytic oxidation of guanine in an intramolecular triplex

Electrocatalytic oxidation of the oligonucleotide 5'- GAA GAG GTT TTT CCT CTT CTT TTT CTT CTC C (TS) by Ru(bpy)(3)(2+) was studied by cyclic voltammetry. This oligonucleotide forms either an intramolecular triplex, hairpin, or single strand, depending on the pH (Plum, G. E.; Breslauer, K. J. J. Mol. Biol. 1995, 248, 679-695). In the triplex form, the guanine doublet in TS is buried inside the folded structure, and as such is less susceptible to oxidation by electrogenerated Ru(bpy)(3)(3+). Digital simulations of the catalytic voltammograms gave a rate constant of 3.5 +/- 0.2 x 10(2) M(-1) s(-1) for oxidation of the triplex form, while oxidation of the duplex and single-stranded forms occurred with much faster rate constants of (3.5-9.1) x 10(4) M(-1) s(-1). Experiments using a truncated form of TS that lacked the third strand of the triplex were consistent with these measurements. The Ru(bpy)(3)(3+) complex was also generated by photolyzing Ru(bpy)(3)(2+) in the presence of Fe(CN)(6)(3-). This reaction produced strand scission following piperidine treatment, which was visualized using high-resolution gel electrophoresis. These experiments showed decreased reactivity for the triplex form, and also gave an unusual reversal of a common selectivity for the 5'-G of GG doublets generally seen in B-form DNA. This reversal was ascribed to strain caused by the location of the GG doublet adjacent to the hairpin loop.     

Assembly of DNA triangles mediated by perylene bisimide caps

Perylene bisimides (PBI) have been synthetically incorporated as caps onto a Y-shaped DNA triple strand. These PBI caps serve as "sticky" ends in the spontaneous assembly of larger DNA ensembles, linking the triangular DNA through stacking interactions. This, in turn, yields a hypsochromic shift in the absorption and a red shift in the fluorescence as characteristic optical readouts. This assembly occurs spontaneously without any enzymatic ligation process and without the use of overhanging DNA as sticky ends. Instead, dimerizations of the PBI chromophores in the assembly are controlled by the DNA as a structural scaffold. Thereby, the PBI-driven assembly is fully reversible. Due to the fact that PBI dimerization does not occur in the single strand, the aggregates can be destroyed by thermal dehybridization of the DNA scaffold and reassembled by reannealing of the DNA construct. In view of the fact that PBI forms stable radical anions, the presented DNA architectures are not only interesting optical biomaterials, but are also promising materials for molecular electronics with DNA.     

Crystal structure of a partly self-complementary peptide nucleic acid (PNA) oligomer showing a duplex-triplex network

The X-ray structure of a partly self-complementary peptide nucleic acid (PNA) decamer (H-GTAGATCACT-l-Lys-NH(2)) to 2.60 A resolution is reported. The structure is mainly controlled by the canonical Watson-Crick base pairs formed by the self-complementary stretch of four bases in the middle of the decamer (G(4)A(5)T(6)C(7)). One right- and one left-handed Watson-Crick duplex are formed. The two PNA units C(9)T(10) change helical handedness, so that each PNA strand contains both a right- and a left-handed section. The changed handedness in C(9)T(10) allows formation of Hoogsteen hydrogen bonding between C(9)T(10) and G(4)A(5) of a PNA strand in an adjacent Watson-Crick double helix of the same handedness. Thereby, a PNA-PNA-PNA triplex is formed. The PNA unit A(3) forms a noncanonical base pair with A(8) in a symmetry-related strand of opposite handedness; the base pair is of the A-A reverse Hoogsteen type. The structural diversity of this PNA demonstrates how the PNA backbone is able to adapt to structures governed by the stacking and hydrogen-bonding interactions between the nucleobases. The crystal structure further shows how PNA oligomers containing limited sequence complementarity may form complex hydrogen-bonding networks.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.