Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate.

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.     

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.     

DNA containing side chains with terminal triple bonds: Base-pair stability and functionalization of alkynylated pyrimidines and 7-deazapurines

The synthesis of a series of oligonucleotides containing 5-substituted pyrimidines as well as 7-substituted 7-deazapurines bearing diyne groups with terminal triple bonds is reported. The modified nucleosides were prepared from the corresponding iodo nucleosides and diynes by the Sonogashira cross-coupling reaction. They were converted into phosphoramidites and employed in solid-phase synthesis of oligonucleotides. The effect of the diyne modifications on the duplex stability was investigated. The modified nucleosides were used for further functionalization using the protocol of Huisgen-Sharpless [2+3] cycloaddition ('click chemistry').     

2-Amino-7-deazaadenine forms stable base pairs with cytosine and thymine.

2-Amino-7-deazaadenine ((AD)A) was incorporated into oligodeoxynucleotides (ODN) and their base-pairing properties with natural nucleobases were investigated. In melting temperature (T(m)) experiments, the duplex containing an (AD)A/C base pair showed a high stability comparable to that containing (AD)A/T base pair. Destabilization of the duplex usually observed for existing degenerate bases was not observed. However, the incorporation efficiency of dCTP was only 1.8% for TTP in single-nucleotide insertion reactions using polymerase.     

Effect of imino group of a linker arm at the C5 position of a pyrimidine nucleoside on the thermal stabilities of DNA/DNA and DNA/RNA duplexes

The modified ODN's bearing C5-substituted 2'-deoxyuridine derivative were synthesized by a post-synthetic modification with an unsymmetrical triamine. The effect of the C5-substituent on the duplex formation with complementary DNA or RNA differed with the position of an imino group in the linker-arms.     

Overcoming potassium-mediated triplex inhibition.

Sequence-specific duplex DNA recognition by oligonucleotide-directed triple helix formation is a possible approach to in vivo gene inhibition. However, triple helix formation involving guanine-rich oligonucleotides is inhibited by physiological ions, particularly K+, most likely due to oligonucleotide aggregation via guanine quartets. Three oligodeoxynucleotide (ODN) derivatives were tested for their ability to resist guanine quartet-mediated aggregation, yet form stable triplexes. Electrophoretic mobility shift and dimethyl sulfate footprinting assays were used to analyze the formation of triplexes involving these oligonucleotide derivatives. In the absence of K+, all ODNs had similar binding affinities for the duplex target. Triplexes involving a 14mer ODN derivative containing 7-deazaxanthine substituted for three thymine bases or an 18mer ODN containing two additional thymines on both the 5' and 3' termini were abolished by 50 mM K+. Remarkably, triplexes involving an ODN derivative containing four 6-thioguanine bases substituted for guanine resisted K+ inhibition up to 200 mM. We hypothesize that the increased radius and decreased electronegativity of sulfur in the 6-position of guanine destabilize potential guanine quartets. These results improve the prospects for creating ODNS that might serve as specific and efficient gene repressors in vivo.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.     

Oligonucleotide duplex stability controlled by the 7-substituents of 7-deazaguanine bases

Oligonucleotides containing 7-substituted 7-deazaguanine residues (7-methyl, 7-iodo) have been synthesized. The self-complemetary octamer d(I⁷c⁷G-C)₄ containing 7-iodo-7-deaza-2′-deoxyguanosine forms a stabilized duplex compared to the parent oligomer d(G-C)₄ (ΔT_m = +10° C). Also the complex between the oligodeoxynucleotide d(I⁷c⁷G₅-G) and poly(C) is stabilized (ΔT_m = +10°) over that of d(c⁷G₅-G) with poly(C).     

Nucleosides and nucleotides. Part 214: thermal stability of triplexes containing 4'alpha-C-aminoalkyl-2'-deoxynucleosides

In order to develop novel antigene molecules forming thermally stable triplexes with target DNAs and having nuclease resistance properties, we synthesized oligodeoxynucleotides (ODNs) with various lengths of aminoalkyl-linkers at the 4'alpha position of thymidine and the aminoethyl-linker at the 4'alpha position of 2'-deoxy-5-methylcytidine. Thermal stability of triplexes between these ODNs and a DNA duplex was studied by thermal denaturation. The ODNs containing the nucleoside 2 with the aminoethyl-linker or the nucleoside 3 with the aminopropyl-linker thermally stabilized the triplexes, whereas the ODNs containing the nucleoside 1 with the aminomethyl-linker or the nucleoside 4 with the 2-[N-(2-aminoethyl)carbamoyl]oxy]ethyl-linker thermally destabilized the triplexes. The ODNs containing 2 were the most efficient at stabilizing the triplexes with the target DNA. The ODNs containing 4'alpha-C-(2-aminoethyl)-2'-deoxy-5-methylcytidine (5) also efficiently stabilized the triplexes with the target DNA. Stability of the ODN containing 5 to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) was studied. It was found that the ODN containing 5 was more resistant to nucleolytic digestion by the enzyme than an unmodified ODN. In a previous paper, we reported that the ODNs containing 2 were more resistant to nucleolytic digestion by DNase I (an endonuclease) than the unmodified ODNs. Thus, it was found that the ODNs containing 4'alpha-C-(2-aminoethyl)-2'-deoxynucleosides were good candidates for antigene molecules.     

The use of oligodeoxynucleotide probes in chaotrope-based hybridization solutions.

Hybridization solutions containing chaotropes may be used to modulate the thermal stability (Tm or Td) of oligodeoxynucleotide (ODN) duplexes or hybrids over a 90 degrees C range. Modulation of Td allows formulation of hybridization solutions that permit ambient temperature hybridization using most combinations of probe length, probe composition, target type, and facilitates development of convenient and rapid assay formats. The conditions required to achieve ODN duplex fidelity, and optimal yields of hybridized product, are described for trichloroacetate, thiocyanate, guanidinium salts and other chaotropic salts. The effects of different solid supports on Td are described. Also, a method is presented that uses chaotropic compounds to reduce background arising from signal ODN probes in a sandwich assay hybridization format.     

Synthesis,thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines

The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (H). The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4–3.9°C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization. Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3′-exonuclease) but also by DNase I (an endonuclease). The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence. Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs. We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H. It was revealed that a minimum of five contiguous unmodified 2′-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H. Thus, the ODN with Ds and at least five contiguous unmodified 2′-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule.     

Reversible photoligation of DNA via 5-vinyldeoxyuridine.

We report a novel non-enzymatic template-directed reversible photoligation of oligodeoxyribonucleotides (ODNs). In this photoligation system, a modified ODN containing 5-vinyldeoxyuridine at 5'-end reacts by photoirradiation at 366 nm with cytosine and thymine at the 3'-end of another ODN in the presence of a complementary template to yield a ligated ODN quantitatively. The ligated ODN can be split site-selectively to regenerate the parent ODN by photoirradiation at 302 nm.     

The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

In an effort to discover novel oligonucleotide modifications for antisense therapeutics, we have prepared oligodeoxyribonucleotides containing more than 200 different modifications and measured their affinities for complementary RNA. These include modifications to the heterocyclic bases, the deoxy-ribose sugar and the phosphodiester linkage. From these results, we have been able to determine structure-activity relationships that correlate hybridization affinity with changes in oligonucleotide structure. Data for oligonucleotides containing modified pyrimidine nucleotides are presented. In general, modifications that resulted in the most stable duplexes contained a heteroatom at the 2'-position of the sugar. Other sugar modifications usually led to diminished hybrid stability. Most backbone modifications that led to improved hybridization restricted backbone mobility and resulted in an A-type sugar pucker for the residue 5'to the modified internucleotide linkage. Among the heterocycles, C-5-substituted pyrimidines stood out as substantially increasing duplex stability.     

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.     

Site-directed alkylation to cytidine within duplex by the oligonucleotides containing functional nucleobases

We have previously described that oligonucleotides (ODN) containing phenylsulfoxide derivative of 2-amino-6-vinylpurine nucleoside analog (1) are activated within duplex to form cross-link toward cytidine selectively at the target site. In this paper, we wish to report the search for more stable precursor susceptible for activation within duplex.     

Cooperative lectin recognition of periodical glycoclusters along DNA duplexes: alternate hybridization and full hybridization

We describe herein the construction of periodically, spatially controlled glycoclusters along DNA duplexes and their cooperative lectin recognition. Site-specifically alpha-mannosylated oligodeoxynucleotide 20-mer (Man-ODN20) was synthesized via the phosphoramidite solid-phase synthesis. Alternate hybridization of the Man-ODN20 with the half-sliding complementary ODN 20-mer (hscODN20) gave an alternately prolonged Man-cluster Man-ODN20/hscODN20. The binding of the Man-cluster to FITC-labeled ConA lectin showed sigmoidal fluorescence dependency on the concentration of Man-ODN, indicating that some mannose residues along the repeating DNA duplex were cooperatively bound to ConA (apparent affinity constant: K(af)=2.4 x 10(4)M(-1) and Hill coefficient: n=3.5). The duplex of Man-ODN20 with full complementary ODN 20-mer (fcODN20) was little bound to ConA. The binding behavior of Man-ODN20/hscODN20 is compared with that of the alternately prolonged Gal-cluster Gal-ODN20/hscODN20 previously reported. Duplexes 20-mer, 40-mer, and 60-mer presenting one, two, and three periodic galactoses were also prepared by full hybridization of 20-mer beta-galactosylated oligodeoxynucleotide (Gal-ODN20) with the periodically repeating full complementary 20-mer, 40-mer, and 60-mer ODNs. RCA(120) lectin was found to little bind the 20-mer and 40-mer duplexes and to bind weakly and non-cooperatively the 60-mer duplex (K(af)=1.1 x 10(4)M(-1)). The cooperative lectin recognition of these glycoclusters in relation with the degree of association (DA) of ODN and the numbers of glycosides along the DNA duplex is discussed.     

化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.