Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.     

Synthesis and DNA Duplex Stabilities of Oligonucleotides Containing C-5-(3-Methoxypropynyl)-2′-deoxyuridine Residues

Abstract

5′-Dimethoxytrityl-5-(3-methoxypropynyl)-2′-deoxyuridine phosphoroamidite was synthesized with the use of commercial 3- methoxypropyne. Oligonucleotides (ODNs) containing 5-(3- ethoxypropynyl)- 2′-deoxyuridine in different positions were prepared. The stabilities of the duplexes formed by these ODNs with the complementary templates are increased in comparison with the unmodified counterparts. On average modified residue incorporated, the Tm is raised by 1°C.     

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.     

Phosphoramidites of (Rp) and (Sp)-Configurated Adenosine Methylphosphonate Dimers

Abstract

The synthesis of adenosine methylphosphonate dimers, the separation of the diastereomers and the conversion into phosphoramidites is presented. These dimer building blocks were tested in the solid phase DNA synthesis of the hairpin decanucleotide 5′-CGCAAAAGCG-3′.     

Intramolecular Recombination R-Triplex in Solution: Stabilization by bis-intercalator YOYO

Abstract

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5′-GT- 2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (R^2APCW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5′- CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R^2APCW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19° C to 33° C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R^2APCW triplex and up to six YOYO molecules to CW hairpin.     

Conformationally Locked Nucleosides. Synthesis of Oligodeoxynucleotides Containing 3′-Amino-3′-deoxy-3′-N,5′(R)-C-ethylenethymidine

Abstract

3′-Amino-3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-N,5′(R)-C-ethylenethymidine (6) was synthesized starting from 3′-azido-3′-deoxythymidine. Condensation of 6 with 5′-O-(H-phosphonyl)thymidine and 5′-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

The First Stereocontrolled Synthesis of Thiooligoribonucleotide: (RpRp)- and (SpSp)-UpsUpsU

Abstract

An approach to the stereocontrolled synthesis of P-homochiral thiooligoribonucleotide: (Rp,Rp)- and (Sp,Sp)-diastereomers of uridinylyl′(3′, 5′)uridinylyl(3′,5′)uridine di (0,0-phosphorothioate) (9) is decribed. The influence of 2′-protection on the efficiency and stereochemistry of the coupling reaction is discussed.     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Preference for Ribose Over Deoxyribose in Loop-Closing Base Pairs of Extra Stable Nucleic Acid Hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5′-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, “extra stable” RNA hairpin structure. Recently, we showed that hairpins containing a 2′,5′-linked (UUCG) loop instead of the native 3′,5′-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368–12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC ₄ and rG ₉ ) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3′,5′-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2′,5′-UUCG loop and, in fact, they may be replaced altogether (ribose → deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.

     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

Hairpin Formation In d-AAGCTTAAGCTT Under High Salt Conditions Shows Unusual Properties

Abstract

The hairpin-duplex equillibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10^?5M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na⁺) for the duplex form is 12 °C and for the hairpin is 18 °C. The ΔH and ΔS values for duplex formation are low(-25 Kcal/mole and—59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4×10⁵M^?1measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCT- AGATCT (Biochemistry 31, 6241–6245) and are discussed in ternis of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.     

Hydration of C-H groups in natural dithymidine nucleotide and its methylphosphonate analogues.

In this paper we report our preliminary studies on the hydration pattern of selected C-H groups in natural thymidyl(3'-5)thymidine and its Rp and Sp-methylphosphonate analogues using Molecular Dynamic simulations in aqueous solutions. The methyl groups attached to the phosphorus center (P-Me) in methylphosphonate analogues are hydrated by water molecules as efficiently as the hydrophilic P=O group in the natural dithymidine nucleotide and better than the neutral P=O functions in these compounds, although the nature of the hydration is different. The C5-Me centers of the 3'-yl units seem to be better hydrated in the methylphosphonate analogues than in the natural dithymidine phosphate and than other centers of the thymine bases in methylphosphonate analogues. Due to chirality of the phosphorus center, the C5-Me group of the 5'-yl unit in the Sp diastereomer coordinates more water than that in the Rp diastereomer. The C6-H group in the 5'-yl unit of the Sp diastereomer exhibits a specific interaction with water.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

An Extended Phosphate Linkage: Synthesis,Hybridization and Modeling Studies of Modified Oligonucleotides

Abstract

Novel stretched oligonucleotides (A-D) containing a 3′-α-C-methylene phosphodiester bridge (5-atoms long) have been synthesized on an automated synthesizer utilizing phosphoramidite chemistry. The key building-block 1-[3′-O-β-cyanoethyldiisopropylaminophosphiryl-2,3-dideoxy-5-O-dimethoxytriphenylmethyl-3-C-(hydroxymethyl)-β-D-erythro-pentofuranosyl]thymine (21) was prepared in a stereoselective manner from thymidine. Hybridization studies indicated a drop (1.8–3.0°C/mod.) in affinity for the complementary RNA and DNA targets. Molecular modeling results indicated that the 5-atom modified backbone had a different geometry around the phosphodiester linkage compared to the natural phosphodiester linkage. The stretched backbone may not be useful for antisense or triplex constructs, however it may find applications in biochemical/enzyme studies.

     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Thermodynamics of melting of the circular dumbbell d〈pCGC-TT-GCG-TT〉

The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson–Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5′-d〈pCGC-TT-GCG-TT〉-3′( I ) and reference compounds 5′-d〈pGGC-TT-GCC-TT≤-3′( II ) and 5′-d(GCG-TC-CGC)-3′( III ) are studied by means of nmr spectroscopy. Molecules I and II adopt dumbbell structures closed on both sides by a two-membered hairpin hop. At low temperature I consists of a mixture of two slowly exchanging forms, denoted L2L2 and L2L4 . The low-temperature L2L2 form is the fully intact minihairpin structure with three Watson–Crick C-G base pairs. The high-temperature form, L2L4 ,contains a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop, with the cytosine base placed in a syn orientation. The opposite 5′-CTTG-3′ loop remains stable. A study of the noncircular hairpin structure III shows similar conformational behavior for the 5′-GTTC-3′ loop as found in I a syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point T_m of II was estimated to lie above 365 K. The T_m value of the duplex stem and the 5′-CTTG-3′ loop of the L2L4 form ofIis 352 ± 2 K. The ΔH° is calculated as ?89 ± 10 kJ/mol. The T_m value determined for the individual residues of the 5′-GTTC-3′ loop lies 4°–11° lower. The enthalpy ΔH° of melting the thymine residues in the 5′-GTTC-3′ loop is calculated to be -61± 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for ΔH° of +30 kJ/mol in going from a two-memberedto a four-membered loop, in agreement with the enthalpy difference of +28 k.j/mol between the two loops at the T_m midpoint. For hairpin III the upper limit for ΔH° going from a two-membered to a four-membered loop amounts to ±21 kJ/mol. The mutual exchange rate between the L2 and L4 form in III is estimated as 13.6 s^?1. Our results clearly suggest that small four-way DNA junctions(model for immobilized Holliday junctions) can be designed that consist of a single DNA strandthat features -CTTG-caps on three of the four arms of the junction. © 1995 John Wiley & Sons, Inc.     

Understanding High Diastereomeric Discrimination in Formation of Oligoribonucleotide Phosphorothioate Linkages: The First Study of pKa-Dependent Activation in Solid-Supported Coupling of 2′-O-Substituted Ribonucleoside Phosphoramidites

Abstract

Activation of 2′-O-substituted ribonucleoside phosphoramidites with various activators during solid-supported synthesis of phosphorothioate oligonucleotides was studied. The Rp:Sp diastereomeric composition of resulting phosphorothioate linkage dependent on pKa of activator utilized for coupling.