Hybridization Properties of RNA Containing 8-Methoxyguanosine and 8-Benzyloxyguanosine

Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt syn conformation of N-glycoside bond. On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogues Containing Novel Phosphotriester Internucleotide Linkage

Abstract

Enhanced cellular uptake, stable and discriminating hybridization and increased stability in biological media are of particular interest for oligonucleotides of potential therapeutic application. Additionally, toxicity or immunogenicity of the oligonucleotide analogues and their biodegradation products should be minimized by minimal alteration of the biological structure and effort and cost of bulk production should be as low as possible by using a standard automated synthesis protocol. Oligonucleotide phosphotriesters with oligoethyleneglycol substituents show promise to ideally combine all these advantages. Here we describe the hybridization properties and the stability of modified oligonucleotides containing triester internucleotide linkages substituted with α,ω-dihydroxy-(3,6-dioxa)-octan-1-yl group (“triethyleneglycol triester linkages”) towards enzymatic degradation. The triester linkages are stable towards exo- and endonucleases. Regardless of number and position of triester linkages, the modified oligonucleotides showed practically no decrease of T_m in hybridization studies with complementary biological oligonucleotides. In further enzymatic studies the modified oligonucleotides were highly stable towards nucleases in human blood serum.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogs Containing Novel Phosphoramidate Internucleotide Linkages

In line with the paradigm, that antisense oligonucleotides should contain minimal structural modifications, in order to minimize the risk of toxicity and antigenicity, we describe here the preparation and the properties of oligonucleotides modified to contain, in addition to phosphodiester bonds, a small number of phosphoramidate internucleotide linkages substituted with aminoethoxyethyl groups in order to convey protection against exo‐ and endonucleases. Prolonged stability was, in fact, found in model experiments with respective enzymes, as well as in studies done in human blood serum. Regardless of number and position of phosphoramidate linkages, the modified oligonucleotides showed only a slight decrease of T_m in hybridization studies with complementary oligonucleotides.     

Hybridization Properties of the 5-Methyl-isocytidine/isoguanosine Base Pair in Synthetic Oligodeoxynucleotides

Abstract

The hybridization properties of the 5-methyl-isocytidine/isoguanosine base pair in synthetic oligodeoxynucleotides have been investigated. The 5-methyl-isocytidine/isoguanosine base pair has been determined to be isoenergetic with the cytidine/guanosine, and each base can effectively discriminate mismatches.     

Chromatographically Fractionated Complementary Strands of Bacillus subtilis Deoxyribonucleic Acid: Biological Properties

Biological, physical, and chromatographic properties of methylated albuminkieselguhr (MAK)-fractionated complementary strands, designated as light (L) and heavy (H), of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. The pattern of transforming activity along the MAK elution profile of alkilidenatured DNA shows that the residually active molecules selectively fractionated ahead of the L strand fraction, whereas the most active self-annealed molecules fractionated preferentially at the trailing end of the H strand fraction. The restoration rate of transforming activity in the late-eluting H molecules was rapid and independent of concentration during the annealing reaction. The data suggest that the self-annealing activity in the H strand is due in part to the formation of intrastrand secondary structures. Hydroxyapatite chromatography of self-annealed L and H strands yielded a major fraction (I) of highly purified strand preparations devoid of transforming activity and hypochromicity, and a minor "nativelike" fraction (II). Sedimentation velocity measurements show that, in addition to the mutual complementary nature of the L and H fractions, they differ in molecular size and possibly configuration.     

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.     

Oligonucleotides Containing a 6-Substituted Pyrimidine Base: A Design for Myb Inhibitors

Abstract

An oligonucleotide having a 6-formylpyrimidine nucleoside in the Myb binding sequence was synthesized based on computer calculation to fit the DNA-protein binding structure.     

4-Guanidino-2-pyrimidinone Nucleobases: Synthesis and Hybridization Properties

Abstract

N-Alkylated 4-guanidino-2-pyrimidinone-containing nucleosides, in which the guanidine group mimics the double hydrogen bond donor pattern of protonated cytosine, were introduced in polypyrimidine sequences to explore their triple-helix forming capabilites. UV and CD melting experiments showed that strands containing these base analogues did not form triplex complexes.     

The Left-Handed Z-DNA Conformation in Oligodeoxynucleotides Containing Different Amounts of AT Base Pairs: A Far UV Circular Dichroism Study

Abstract

A number of fully self-complementary oligodeoxynucleotides have been synthesized and examined for their ability to assume the left-handed Z-DNA conformation in high salt solutions. The B- and Z-forms are identified by circular dichroism spectra, covering both the long-(220–300 nm) and short-wavelength (185–220 nm) regions, the latter showing CD bands very useful for identifying the sense of the helix winding. The main results of the study can be summarized as follows:

a) sequences composed by AT and CG blocks do support the B to Z transition, even when the AT contents amounts to 50%;

b) the occurrence of consecutive purine-purine or pyrimidine-pyrimidine dyads does not inhibit the B to Z transition, although a stronger reduction of water activity is required;

c) (AC)_n and (GT)_n containing oligonucleotides do undergo the B to Z transition in solution;

d) a millimolar quantity of Ni²⁺ concomitant with 5 M NaC10₄ is found to be very effective in bringing about the B to Z transition in most of the sequences considered in this study.     

Synthesis and Characterization of Oligodeoxyribonucleotides Containing Tandem Base Damage

Abstract

Both N(2-deoxy-β-D-erythro-pentofuranosyl)-formylamine (dβF) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) were introduced in synthetic oligonucleotides at a vicinal position via the solid phase phosphoramidite method in order to investigate the biological and structural significance of such a tandem lesion. Further experiments aimed at determining the enzymatic repair by both E. coli endonuclease III (Endo III) and Fapy-glycosylase (Fpg) were carried out with these synthetic substrates.     

Synthesis and Hybridization Properties of Oligonucleotides Containing (2 S ,3 R )-9-(2,3,4-Trihydroxybutyl)Adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A ^C2 and A ^C3, are described. The ON containing A ^C2 involves the 3′ → 4′ and 3′ → 5′ phosphodiester linkages in the strand, whereas that containing A ^C3 possesses the 3′ → 4′ and 2′ → 5′ phosphodiester linkages. It was found that incorporation of the analogs, A ^C2 or A ^C3, into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A ^C2 is greater than that of A ^C3 in the ON/RNA duplexes.     

Phosphonate Analogues of Peptide Nucleic Acids and Related Compounds: Synthesis and Hybridization Properties

Abstract

The synthesis of a set of novel chimeric oligomers representing PNA and phosphono-PNA analogues has been accomplished, and their binding affinity to complementary DNA and RNA strands was evaluated.     

Synthesis and Properties of DNA Containing Cyclonucleosides

Here, we present efficient syntheses of the R and S diastereomers of 8,5′-cyclo-2′-deoxyadenosine and 6,5′-cyclo-2′-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.     

The Functions of Atrial Strands Interdigitating with and Penetrating into Sinoatrial Node: A Theoretical Study of the Problem

The sinoatrial node (SAN)-atrium system is closely involved with the activity of heart beating. The impulse propagation and phase-locking behaviors of this system are of theoretical interest. Some experiments have revealed that atrial strands (ASs) interdigitate with and penetrate into the SAN, whereby the SAN-atrium system works as a complex network. In this study, the functions of ASs are numerically investigated using realistic cardiac models. The results indicate that the ASs penetrating into the central region of the SAN play a major role in propagating excitation into the atrium. This is because the threshold SAN-AS coupling for an AS to function as an alternative path for propagation is lower at the center than at the periphery. However, ASs penetrating into the peripheral region have a great effect in terms of enlarging the 1:1 entrainment range of the SAN because the automaticity of the SAN is evidently reduced by ASs. Moreover, an analytical formula for approximating the enlargement of the 1:1 range is derived.     

Crystal Structure of A Z-DNA Fragment Containing Thymine/2-Aminoadenine Base Pairs

Abstract

The Z-DNA structure has been shown to form in two crystals made from self-complementary DNA hexamers d(CGTDCG) and d(CDCGTG) which contain thymine/2-ammoadenine (TD) base pairs. The latter structure has been solved and refined to 1.3 Å resolution and it shows only small conformational changes due to the introduction of the TD base pairs in comparison with the structure of d(CG)3. Spectroscopic studies with these compounds demonstrate that DNA molecules containing 2-aminoadenine residues form Z-DNA slightly more easily than do those containing adenine nucleotides, but not as readily as the parent sequence containing only guanine-cytosine base pairs.     

Characterization and Genome Changes of New Amphiploids from Wheat Wide Hybridization

<正>The introduction of alien genes or chromosome fragments from wild related species into wheat(Triticum spp.)has been considered valuable for wheat breeding(Valkoun,2001).Several relatives,such as Secale cereale(Gupta and Shepherd,1993;Ma et al.,2000),Thinopyrum elongatum(Liu et al.,2008),Th.intermedium(Cao et al.,2014)and Th.ponticum(Li et al.,2008)showed distinct applications of introgression of interesting genes or traits into wheat.However,there are still some challenges in using these genetic resources.The     

红菜薹与甘蓝型油菜杂交子房培养研究初报

对甘蓝型油菜和红菜薹种间杂种进行胚胎挽救研究,实验结果表明,采用MS＋（1．0～2．0）mg·L^-1 6-BA＋0．05mg·L^-1 NAA＋0．5％活性炭＋30g·L^-1蔗糖＋7．5g·L^-1琼脂培养基对甘蓝型油菜和红菜薹杂交子房培养效果较为理想;相对于培养基和激素,活性炭对子房培养的影响更加显著。通过对取材时间的研究发现,取授粉后18d子房培养的结籽率最高,15d的次之。而通过对杂种萌发率的研究则表明,授粉后15d的子房培养获得的杂种萌发率最高,为57．03％,18d的最差,仅为38．49％。     

Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.