Reactive derivatives of oligonucleotide phosphorothioate analogues: IV. Site-directed modification of nucleic acids and intramolecular alkylation of phosphorothioate groups

Alkylation of the 22-mer DNA target pTGCCTGGAGCTGCTTGATGCCC (I) by oligodeoxynucleotide phosphorothioate derivatives (PTAO) GpsCpsApsTpsCpsApsApsGpsCpsApsGpsCpN(CH₃)CH₂(RCl)(II-PS) and (RCl)CH₂N(CH₃)pGpsCpsAps TpsCpsApsApsGpsCpsApsGpsC (III-PS) bearing a residue of an aromatic analogue of nitrogen lost (RCl=C₆H₄N(CH₃)(CH₂CH₂Cl) at the 3′- or 5′-end was studied. It was shown that the internucleotide phosphorothioate bonds do not affect the regiospecificity of the target modification. The maximum degree of the target modification (att→∞) at 20°C was about 25% for both (II-PS) and (III-PS). The use of GCATCAAGCAGCpN(CH₃)CH₂(RCl)(II-PO), containing internucleotide phosphodiester bonds, under the same conditions gave about 65% of the modified DNA. Kinetics of the PTAO-induced complementarily addressed nucleic acid (NA) modification was analyzed. The rate constants of the reaction of the intermediate reactive ethylenimmonium ion with phosphorothioate groups of the reagents were evaluated both in solution and in duplex. The intramolecular alkylation of phosphorothioate groups considerably affected the DNA target modification by decreasing the effectiveness of the modification in a wide range of temperatures and changing the temperature dependence of the modification from a bell-like to an S-like profile. It was concluded that, in the course of the modification, the PTAO phosphorothioate groups are intramolecularly alkylated both in solution and in the complementary NA target-oligonucleotide duplex. For Part III, see [1].     

Reactivity of oligonucleotide derivatives, containing methylphosphonate groups. VI. Increase in the effectiveness of directed action of alkylating derivatives of oligonucleotide methylphosphonate analogs on nucleic acids in the presence of effectors--3',5'-bis-N-(2-hydroxyethyl)-phenazine derivatives of oligonucleotides

Effectors for increasing the efficiency of DNA modification with the alkylating methylphosphonate analogues of oligodeoxyribonucleotides (MFAO) were suggested. Oligodeoxyribonucleotide d(pC5A8ACAATG) used as a target DNA treated with alkylating derivatives of octathymidylate having alternating methylphosphonate and phosphodiester internucleotide bonds (both Rp- and Sp-individual diastereoisomers of MFAO were used) and bearing alkylating 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide residue at the 3'-end. The reactions were carried out in the presence of an effector, hexadeoxyribonucleotide derivative PhnNH(CH2)2NHpCATTGTpNH(CH2)2NHPhn bearing two N-(2-hydroxyethyl)phenazinium (Phn) residues at the 3'- and 5'-ends and being complementary to the part of the target DNA neighbouring with octaadenylate. It was shown that Tm of the duplex formed by the target DNA, octathymidylate and effector is by 7-13 degrees C higher than in the absence of the effector, thus considerably increasing the efficiency of the intracomplex alkylation of the target (e.g., at 40 degrees C, the increase for the reagent based on the Rp-isomer is sixfold). Specificity of the target DNA modification by the MFAO alkylating derivatives in the presence of effector is same as with reagents based on oligodeoxyribonucleotides with natural internucleotide bonds.     

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.     

Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.     

Selective binding of trisamine-modified phosphorothioate antisense DNA to target mRNA improves antisense activity and reduces toxicity

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature (T(m)) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.     

Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A

The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products.     

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.     

Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers.

The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations.     

Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics

DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.     

Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.

Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity. For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.     

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues.

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.     

Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity.     

Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.     

Site specific functionalization of oligonucleotides for attaching two different reporter groups.

The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.     

Absolute configuration of Rp-uridine 3'',5''-cyclic phosphorothioate.

Triethylammonium uridine-3',5'-cyclic phosphorothioate crystallizes in space group P2(1)2(1)2(1), a = 7.177(1), b = 13.155(6), c = 21.114(7) A, C15H26N3O7PS, MW 423.4, Z = 4, dx = 1.41g/cm3. The crystal structure was solved by direct methods on the basis of 1493 counter X-ray diffraction data (CuK alpha) and refined to R = 5.1%. The configuration of the thiophosphate group is Rp; conformational parameters are: glycosyl torsion angle anti, -151.9(5) degrees, sugar pucker C(3')-endo with P = 27.3 degrees, vmax = 45.5 degrees, six-membered cycle in chair form. The bond distances in the non-esterified P-S and P-O suggest that the negative charge is distributed between the groups. As illustrated in this and other studies, P-O has a much higher affinity for hydrogen bonds than P-S, indicated here by interactions with triethyl-ammonium N-H and O(2')-H as donors. One additional hydrogen bond N(3)-H---0(4) ties the bases which form a ribbon-like structure. 0(2) and S are not engaged in hydrogen bonds. The triethylammonium ion is two-fold disordered.     

Influence of a Thiophosphate Linkage on the Duplex Stability - Does Sp Configuration Always Lead to Higher Stability Than Rp?

Abstract

ABSTRACT: In order to design an oligodeoxynucleoside phosphorothioate as an antisense molecule, it is important to establish the structure of the S-oligo with a strong affinity to the target RNA. In these molecules, internucleotide thiophosphate linkages produce diastereomers, the number of which increases in proportion to 2ⁿ (n: number of thiophosphate). To estimate the effect of this linkage on the duplex stability by UV melting curves, oligodeoxynucleotides having a single thiophosphate (referred to Soligo), dGCNsN'CG (s: thiophosphate, N, N′ = A or T), were prepared and their diastereomers isolated by HPLC. As demonstrated previously, the melting temperatures (Tm) for the Sp isomers were higher than those of the Rp when DNA was a target. On the other hand, it was found that for RNA as a target, the Rp isomers of dGCTsTCG and dGCAsTCG had higher stability than the Sp, and that the difference in the Tm values between the diastereomers was smaller than when DNA was a target. With dGCsTsACG, which has two thiophosphates, it was also found that the Tm values decreased with an increase in the number of thiophosphate linkages, and that the difference in Tm between the diastereomers was smaller when RNA was a target. Consequently, in practical clinical applications where RNA is a target, the influence of thiophosphate chirality on the duplex structure is almost negligible and Rp/Sp separation of an S-oligo may be of no major concern.     

Comparison of the cleavage profiles of oligonucleotide duplexes with or without phosphorothioate linkages by using a chemical nuclease probe

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO₅ is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5-GCAAAAGC/5-GCTTTTGC duplexes by Mn-TMPyP/KHSO₅ was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all-Rp-phosphorothioate and all-Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)₃-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

基因组磷硫酰化修饰的研究进展及展望北大核心CSCD

DNA磷硫酰化修饰是DNA骨架上的第一例生理修饰。该修饰由dnd ABCDE编码的5个蛋白协同作用,以硫原子取代DNA磷酸二酯键上一个非桥接的氧原子。研究发现,DNA磷硫酰化修饰广泛存在于各种微生物中,在不同细菌中存在序列特异性,且具有R_P空间构象专一性。近年来,对DNA磷硫酰化修饰的研究取得了一系列的成果。为了对DNA磷硫酰化修饰有一个系统全面的了解,本文将就这一特殊生理修饰的发现过程,研究进展,未来所面临的机遇及挑战作一个简要的概述。