Comparison of the RNase H cleavage kinetics and blood serum stability of the north-conformationally constrained and 2'-alkoxy modified oligonucleotides.

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1',2'-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2',4'-ethylene-bridged (aza-ENA-T) nucleoside, and 2'-alkoxy modified nucleosides (2'-O-Me-T and 2'-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (DeltaTm = 2.5-4 degrees C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5 degrees C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of approximately 5-6 degrees C per modification. The 2'-O-Me-T and 2'-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (approximately 1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (approximately 1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (approximately 1.1-3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2'-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2'-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3'-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3'-exonuclease preferentially hydrolyzed the 3'-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3'-exonuclease cleaved at the 3'-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5'-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5'-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2'-O-Me-T, and 2'-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

Oxetane locked thymidine in the Dickerson-Drew dodecamer causes local base pairing distortions -- an NMR structure and hydration study.

The introduction of a North-type sugar conformation constrained oxetane T block, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl) thymine, at the T(7) position of the self-complementary Dickerson-Drew dodecamer, d[(5'-C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12)-3')](2), considerably perturbs the conformation of the four central base pairs, reducing the stability of the structure. UV spectroscopy and 1D NMR display a drop in melting temperature of approximately 10 degrees C per modification for the T(7) oxetane modified duplex, where the T(7) block has been introduced in both strands, compared to the native Dickerson-Drew dodecamer. The three dimensional structure has been determined by NMR spectroscopy and has subsequently been compared with the results of 2.4 ns MD simulations of the native and the T(7) oxetane modified duplexes. The modified T(7) residue is found to maintain its constrained sugar- and the related glycosyl torsion conformations in the duplex, resulting in staggered and stretched T(7).A(6) and A(6).T(7) non-linear base pairs. The stacking is less perturbed, but there is an increased roll between the two central residues compared to the native counterpart, which is compensated by tilts of the neighboring base steps. The one dimensional melting profile of base protons of the T(7) and T(8) residues reveals that the introduction of the North-type sugar constrained thymine destabilizes the core of the modified duplex, promoting melting to start simultaneously from the center as well as from the ends. Temperature dependent hydration studies by NMR demonstrate that the central T(7).A(6)/A(6).T(7) base pairs of the T(7) oxetane modified Dickerson-Drew dodecamer have at least one order of magnitude higher water exchange rates (correlated to the opening rate of the base pair) than the corresponding base pairs in the native duplex.

A uniform mechanism correlating dangling-end stabilization and stacking geometry.

The geometry of the dangling base in 105 published structures (from X-ray/NMR) containing single-stranded overhangs has been analyzed and correlated to the thermodynamic stabilization found (UV) for the corresponding dangling base/closing basepair combination in short oligonucleotides. The study considers most combinations of closing basepairs, sequence and dangling base residue type, attached in both the 3'- and 5'-ends of both DNA and RNA. Linear regression analysis showed a straightforward correlation (R = 0.873) between the degree of screening for the hydrogen bonds of the closing basepair provided by the dangling base and the resulting thermodynamic stabilization in both DNA and RNA series with dangling ends either at the 3'- or at the 5'-terminus. Regression analysis of only the datasets from RNA gives an improved correlation, R = 0.934, showing that dangling ends on RNA are more ordered than the dangling ends on DNA, R = 0.376. This study highlights the gain in the free energy of stabilization owing to the favorable stacking between the dangling nucleobase and the neighboring basepair and the resulting strengthening of the hydrogen bond of the closing basepair. By acting as a hydrophobic cap on the terminal of the DNA or RNA duplex, the dangling-end residue restricts the bulk water access to the terminal basepair, thereby providing it with a microenvironment devoid of water, which consequently enhances its thermodynamic stability, making it energetically comparable to the corresponding internal basepair. Thus, one single structural model consisting of the interplay of the above electrostatic interactions can be used to explain the molecular basis of the observed thermodynamic effects for dangling-end attachment to the 3'- and 5'-ends of both DNA and RNA duplexes, which is a key step toward accurate dangling-end effect prediction.

Single-stranded adenine-rich DNA and RNA retain structural characteristics of their respective double-stranded conformations and show directional differences in stacking pattern.

The structural preorganization of isosequential ssDNA and ssRNA hexamers d/r(GAAAAC)(1) [J. Am. Chem. Soc. 2003, 125, 9948] have been investigated by NMR and molecular dynamics simulations. Analysis of the nuclear Overhauser effect spectrometry (NOESY) footprints in the aqueous solution has shown that there is a substantial population of ordered right-handed helical structure in both hexameric single-stranded DNA and RNA, which are reminiscent of their respective right-handed helical duplex form, despite the fact these single-stranded molecules are devoid of any intermolecular hydrogen bonds. The NMR-constrained molecular dynamics (1.5 ns) derived geometries of the adenine-adenine overlaps at each dinucleotide step of the hexameric ssDNA (1a) and ssRNA (1b) show that the relatively electron-rich imidazole stacks above the electron-deficient pyrimidine in 5' to 3' direction in ssDNA (1a) while, in contradistinction, the pyrimidine stacks above the imidazole in the 5' to 3' direction in ssRNA (1b). This also means that the pi-frame of the 5'-pyrimidine can interact with the relatively positively charged imino and amino protons in the 3' direction in ssRNA and in the 5' direction in ssDNA, thereby stabilizing the twist and slide observed in the stacked oligonucleotides. The differently preferred stacking geometries in ssDNA and ssRNA have direct physicochemical implications for self-assembly and pK(a) modulation by the nearest-neighbor interactions, as well as for the dangling-end stabilization effects and imino-proton reactivity.

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Measurement of nucleobase pKa values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes.

To understand why the RNA-RNA duplexes in general has a higher thermodynamic stability over the corresponding DNA-DNA duplexes, we have measured the pK(a) values of both nucleoside 3',5'-bis-ethyl phosphates [Etp(d/rN)pEt] and nucleoside 3'-ethyl phosphates [(d/rN)pEt] (N = A, G, C, or T/U), modeling as donors and acceptors of base pairs in duplexes. While the 3',5'-bis-phosphates, Etp(d/rN)pEt, mimic the internucleotidic monomeric units of DNA and RNA, in which the stacking contribution is completely absent, the 3'-ethyl phosphates, (d/rN)pEt, mimic the nucleotide at the 5'-end. The pK(a) values of the nucleobase in each of these model nucleoside phosphates have been determined with low pK(a) error (sigma = +/-0.01 to 0.02) by (1)H NMR (at 500 MHz) with 20-33 different pH measurements for each compound. This study has led us to show the following: (1) All monomeric DNA nucleobases are more basic than the corresponding RNA nucleobases in their respective Etp(d/rN)pEt and (d/rN)pEt. (2) The pK(a) values of the monomeric nucleotide blocks as well as Delta pK(a) values between the donor and acceptor can be used to understand the relative base-pairing strength in the oligomeric duplexes in the RNA and DNA series. (3) The Delta G*(pKa) of the donor and acceptor of the base pair in duplexes enables a qualitative dissection of the relative strength of the base-pairing and stacking in the RNA-RNA over the DNA-DNA duplexes. (4) It is also found that the relative contribution of base-pairing strength and nucleobase stacking in RNA-RNA over DNA-DNA is mutually compensating as the % A-T/U content increases or decreases. This interdependency of stacking and hydrogen bonding can be potentially important in the molecular design of the base-pair mimics to expand the alphabet of the genetic code.

Cross-modulation of the pKa of nucleobases in a single-stranded hexameric-RNA due to tandem electrostatic nearest-neighbor interactions.

The pH titration studies (pH 6.7-12.1) in a series of dimeric, trimeric, tetrameric, pentameric, and hexameric oligo-RNA molecules [GpA (2a), GpC (3a), GpApC (5), GpA(1)pA(2)pC (6), GpA(1)pA(2)pA(3)pC (7), GpA(1)pA(2)pA(3)pA(4)pC (8)] have shown that the pK(a) of N(1)-H of 9-guaninyl could be measured not only from its own deltaH8G, but also from the aromatic marker protons of other constituent nucleobases. The relative chemical shift differences [Deltadelta((N)(-)(D))] between the protons in various nucleotide residues in the oligo-RNAs at the neutral (N) and deprotonated (D) states of the guanine moiety show that the generation of the 5'-(9-guanylate ion) in oligo-RNAs 2-8 reduces the stability of the stacked helical RNA conformation owing to the destabilizing anion(G(-))-pi/dipole(Im(delta)(-)) interaction. This destabilizing effect in the deprotonated RNA is, however, opposed by the electrostatically attractive atom-pisigma (major) as well as the anion(G(-))-pi/dipole(Py(delta)(+)) (minor) interactions. Our studies have demonstrated that the electrostatically repulsive anion(G(-))-pi/dipole(Im(delta)(-)) interaction propagates from the first to the third nucleobase quite strongly in the oligo-RNAs 6-8, causing destacking of the helix, and then its effect is gradually reduced, although it is clearly NMR detectable along the RNA chain. Thus, such specific generation of a charge at a single nucleobase moiety allows us to explore the relative strength of stacking within a single-stranded helix. The pK(a) of 5'-Gp residue from its own deltaH8G in the hexameric RNA 8 is found to be 9.76 +/- 0.01; it, however, varies from 9.65 +/- 0.01 to 10.5 +/- 0.07 along the RNA chain as measured from the other marker protons (H2, H8, H5, and H6) of 9-adeninyl and 1-cytosinyl residues. This nucleobase-dependent modulation of pK(a)s (DeltapK(a) +/- 0.9) of 9-guaninyl obtained from other nucleobases in the hexameric RNA 8 represents a difference of ca. 5.1 kJ mol(-)(1), which has been attributed to the variable strength of electrostatic interactions between the electron densities of the involved atoms in the offset stacked nucleobases as well as with that of the phosphates. The chemical implication of this variable pK(a) for guanin-9-yl deprotonation as obtained from all other marker protons of each nucleotide residue within a ssRNA molecule is that it enables us to experimentally understand the variation of the electronic microenvironment around each constituent nucleobase along the RNA chain in a stepwise manner with very high accuracy without having to make any assumption. This means that the pseudoaromaticity of neighboring 9-adeninyl and next-neighbor nucleobases within a polyanionic sugar-phosphate backbone of a ssRNA can vary from one case to another due to cross-modulation of an electronically coupled pi system by a neighboring nucleobase. This modulation may depend on the sequence context, spatial proximity of the negatively charged phosphates, as well as whether the offset stacking is ON or OFF. The net outcome of this electrostatic interaction between the neighbors is creation of new sequence-dependent hybrid nucleobases in an oligo- or polynucleotide whose properties are unlike the monomeric counterpart, which may have considerable biological implications.

The pK(a) of the internucleotidic 2'-hydroxyl group in diribonucleoside (3'-->5') monophosphates.

Ionization of the internucleotidic 2'-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariat-RNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pK(a) of 2'-OH in mono- and diribonucleoside (3'-->5') monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pK(a) values for internucleotidic 2'-OH of eight different diribonucleoside (3'-->5') monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pK(a) is 12.31 (+/-0.02) for ApG and 12.41 (+/-0.04) for ApA, 12.73 (+/-0.04) for GpG and 12.71 (+/-0.08) for GpA, 12.77 (+/-0.03) for CpG and 12.88 (+/-0.02) for CpA, and 12.76 (+/-0.03) for UpG and 12.70 (+/-0.03) for UpA. By comparing the pK(a)s of the respective 2'-OH of monomeric nucleoside 3'-ethyl phosphates with that of internucleotidic 2'-OH in corresponding diribonucleoside (3'-->5') monophosphates, it has been confirmed that the aglycons have no significant effect on the pK(a) values of their 2'-OH under our measurement condition, except for the internucleotidic 2'-OH of 9-adeninyl nucleotide at the 5'-end (ApA and ApG), which is more acidic by 0.3-0.4 pK(a) units.

Tandem electrostatic effect from the first to the third aglycon in the trimeric RNA owing to the nearest-neighbor interaction.

We here show an electrostatic polar-pi interaction from the first to the third aglycon, via the second aglycon, in the ground state in two single stranded trimeric RNAs, 5'-GpA(1)pA(2)-3' (3) and 5'-GpApC-3' (4), as a result of intramolecular nearest neighbor offset-stacking. The experimental evidence in support of this conclusion has been obtained by comparing the pK(a)s of each aglycone in the two trimers with those of guanosine 3'-ethyl phosphate, GpEt (1) and 5'-GpA-3' (2): Thus, the pK(a) of N(1)-H of guanin-9-yl of 5'-GpA(1)pA(2)-3' (3) could be measured by pH titration (pH 7.3-11.6) of its own deltaH8G (pK(a) 9.75 +/- 0.02) as well as from deltaH8A(1) (pK(a) 9.72 +/- 0.02) and deltaH2A(1) (pK(a) 9.83 +/- 0.04) of the neighboring pA(1)p moiety and the deltaH8A(2) (pK(a) 9.83 +/- 0.02) of the terminal pA(2) moiety. Similarly, the pH titration of GpApC (4) shows the pK(a) of N(1)-H of guanin-9-yl from its own deltaH8G (pK(a) 9.88 +/- 0.03) as well as from deltaH8A (pK(a) 9.87 +/- 0.01) of the neighboring pAp moiety, and deltaH5/H6C (pK(a) 9.88 +/- 0.01 and 9.90 +/- 0.01 respectively) of the 3'-terminal cytosin-1-yl. This intramolecular nearest neighbor electrostatic interaction in the single-stranded RNA modulates the pseudoaromaticity of the nearest neighbors by almost total transmission of because they constitute an extended array of offset-stacked coupled aromatic heterocycles within a polyanionic sugar-phosphate backbone at the ground state. The enhanced basicity of Gp residue by ca. 0.6 pK(a) unit in the trimers compared to that of the dimer is a result of the change in the electrostatic microenvironment owing to the nearest neighbors in the former (the nucleobases as well as the phosphates). Thus, the from the 5'-guanylate ion to the 3'-end aglycon via the central adenin-9-yl is 55 to 56 kJ mol(-)(1) in each step through a distance spanning approximately 6.8 A in an unfolded state. As a result, the pK(a) of guanin-9-yl moiety has become 9.25 +/- 0.02 in GpEt (1), 9.17 +/- 0.02 in GpA (2), 9.75 +/- 0.02 in GpApA (3), and 9.88 +/- 0.03 in GpApC (4). This means that guanin-9-yl moiety of trimers 3 and 4 is more basic than in the monomer or the dimer. The net outcome of this electrostatic cross-talk between the two neighboring heterocycles is creation of new hybrid aglycones in an oligo or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the nearest neighbors, and whether they are stacked or unstacked. Thus, this tunable physicochemical property of an aglycon (an array of the extended genetic code) may have considerable implication in our understanding of the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability of nucleic acids in a microenvironment, or in the triplet usage by the anticodon-codon interaction in the protein biosynthesis in the ribosome.

A repertoire of pyridinium-phenyl-methyl cross-talk through a cascade of intramolecular electrostatic interactions.

Direct intramolecular cation-pi interaction between phenyl and pyridinium moieties in 1a(+) has been experimentally evidenced through pH-dependent (1)H NMR titration. The basicity of the pyridinyl group (pK(a) 2.9) in 1a can be measured both from the pH-dependent chemical shifts of the pyridinyl protons as well as from the protons of the neighboring phenyl and methyl groups as a result of electrostatic interaction between the phenyl and the pyridinium ion in 1a(+) at the ground state. The net result of this nearest neighbor electrostatic interaction is that the pyridinium moiety in 1a becomes more basic (pK(a) 2.92) compared to that in the standard 2a (pK(a) 2.56) as a consequence of edge-to-face cation (pyridinium)-pi (phenyl) interaction, giving a free energy of stabilization (DeltaDeltaG(o)pKa) of -2.1 kJ mol(-1). The fact that the pH-dependent downfield shifts of the phenyl and methyl protons give the pK(a) of the pyridine moiety of 1a also suggests that the nearest neighbor cation (pyridinium)-pi (phenyl) interaction also steers the CH (methyl)-pi (phenyl) interaction in tandem. This means that the whole pyridine-phenyl-methyl system in 1a(+) is electronically coupled at the ground state, cross-modulating the physicochemical property of the next neighbor by using the electrostatics as the engine, and the origin of this electrostatics is a far away point in the molecule-the pyridinyl-nitrogen. The relative chemical shift changes and the pK(a) differences show that the cation (pyridinium)-pi (phenyl) interaction is indeed more stable (DeltaDeltaG(o)pKa = -2.1 kJ mol(-1)) than that of the CH (methyl)-pi (phenyl) interaction (DeltaDeltaG(o)pKa = -0.8 kJ mol(-1)). Since the pK(a) of the pyridine moiety in 1a is also obtained through the pH-dependent shifts of both phenyl and methyl protons, it suggests that the net electrostatic mediated charge transfer from the phenyl to the pyridinium and its effect on the CH (methyl)-pi (phenyl) interaction corresponds to DeltaG(o)pKa of the pyridinium ion (approximately 17.5 kJ mol(-1)), which means that the aromatic characters of the phenyl and the pyridinium rings in 1a(+) have been cross-modulated owing to the edge-to-face interaction proportional to this DeltaG(o)pKa change.

Cross-modulation of physicochemical character of aglycones in dinucleoside (3'-->5') monophosphates by the nearest neighbor interaction in the stacked state.

Each nucleobase in a series of stacked dinucleoside (3'-->5') monophosphates, in both acidic and alkaline pH, shows ((1)H NMR) not only its own pK(a) but also the pK(a) of the neighboring nucleobase as a result of cross-modulation of two-coupled pi systems of neighboring aglycones. This means that the electronic character of two nearest neighbors are not like the monomeric counterparts anymore; they have simultaneously changed, almost quantitatively, to something that is a hybrid of the two due to two-way transmission of charge (i.e. 3'-->5' as well as 5'-->3'). This change is permanent due to total modulation of each others pseudoaromatic character by intramolecular stacking, which can be tuned by the nature of the medium across the whole pH range. The small difference observed in the pK(a) of the dimer compared to the monomer is a result of the change in microenvironment in the former. The charge transfer takes place between two stacked nucleobases from the negatively charged end because of the attempt to minimize the charge difference between the two neighboring pseudoaromatic aglycones. Experimental evidence points that the charge transmission in the stacked state takes place by atom-pisigma interaction between nearest neighbor nucleobases in 1-6. The net result of this cross-talk between two neighboring aglycones is a unique set of aglycones in an oligo- or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the sequence makeup, and whether they are stacked or unstacked. Thus, the physicochemical property of individual nucleobases in an oligonucleotide is determined in a tunable manner, depending upon who the nearest neighbors are, which may have considerable implication in the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability in a microenvironment, in the use of codon triplets in the protein biosynthesis or in the triplet usage by the anticodon-codon interaction.

Total synthesis of 2',3',4',5',5' '-(2)h(5)-ribonucleosides: the key building blocks for NMR structure elucidation of large RNA.

The diastereospecific chemical syntheses of uridine-2',3',4',5',5' '-(2)H(5) (21a), adenosine-2',3',4',5',5' '-(2)H(5) (21b), cytidine-2',3',4',5',5' '-(2)H(5)(2)H(5) (21c), and guanosine-2',3',4',5',5' '-(2)H(5) (21d) (>97 atom % (2)H at C2', C3', C4', and C5'/C5' ') have been achieved for their use in the solution NMR structure determination of oligo-RNA by the Uppsala "NMR-window" concept (refs 4a-c, 5a, 6), in which a small (1)H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the deuterated blocks 21a-d. The deuterated ribonucleosides 21a-d have been prepared by the condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-alpha/beta-D-ribofuranose-2,3,4,5,5'-(2)H(5) (19), which has been obtained via diastereospecific deuterium incorporation at the C2 center of appropriate D-ribose-(2)H(4) derivatives either through an oxidation-reduction-inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%, respectively).

An energetic correlation of ab initio and NMR studies of the 3'-gauche effect in 3'-substituted thymidines.

A straightforward correlation of our experimental NMR findings on 3'-substituted thymidine derivatives with that of the ab initio calculations shows that (i) the delta Go298kNRM of N reversible S equilibrium in nucleoside can be predicted from the ab initio calculated delta ES-N obtained from 6-311++G** level of theory; (ii) the substituent-dependent steric and stereoelectronic effects on the bias of the two-state N reversible S equilibrium in nucleosides can also be predicted from the ab initio calculations with sufficiently large basis functions, and (iii) the necessity of mimicking the solvation behaviour of the experimental NMR measurement condition in the ab initio calculations of biomolecules is also emphasized.

Molecular modelling of 2'-OH mediated hydrogen bonding in ribonucleos(t)ides by NMR constrained AM1 and MMX calculations.

The intra- and intermolecular hydrogen bonding (delta Go298K approximately 2 kcal mol-1) of 2'-OH in nucleos(t)ides has been reported by the temperature- and concentration-dependent NMR study in conjunction with dihedral dependence of the NMR derived both endo (3JH,H)- and exocyclic (3JH,OH) coupling constants, nOe contacts and lineshape analyses of hydroxyl protons for EtpA (1), 3'-dA (2), rA (3), 2'-dA (4) [Fig. 1] in DMSO-d6 at 500 MHz.

Synthetic studies to improve the deuterium labelling in nucleosides for facilitating structural studies of large RNAs by high-field NMR spectroscopy.

Synthetic studies to prepare ribonucleosides deuterated at C2' and the application of the developed procedures for the synthesis of 2H5-ribonucleosides from 1,2-O-isopropylidene-3-O-benzyl-ribofuranose-3,4,5,5'-2H4 have been reported.

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

Synthesis of [Ru(phen)(2)dppz](2+)-tethered oligo-DNA and studies on the metallointercalation mode into the DNA duplex.

To explore the binding properties of [Ru(phen)(2)dppz](2+) complex (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) in a sequence-specific manner in DNA duplex, it was tethered through the dppz ligand to a central position as well as both at the 3'- and 5'-ends of oligodeoxyribonucleotide (ODN). The middle [Ru(phen)(2)dppz](2+)-ODN tethered was resolved and isolated as four pure diastereomers, while the 3'- or 5'-[Ru(phen)(2)dppz](2+)-ODNs were inseparable on RP-HPLC. Thermal stability of the (Ru(2+)-ODN).DNA duplexes is found to increase considerably (DeltaT(m) = 12.8-23.4 degrees C), depending upon the site of the covalent attachment of the tethered [Ru(phen)(2)dppz](2+) complex, or the chirality of the [Ru(phen)(2)dppz](2+)-linker tethered at the middle of the ODN, compared to the unlabeled counterpart. Gross differences in CD between the [Ru(phen)(2)dppz](2+)-tethered and the native DNA duplexes showed that the global duplex conformation of the former has considerably altered from the B-type, but is still recognized by DNase I. The thermal melting studies, CD measurements, as well as DNase I digestion data, are interpreted as a result of intercalation of the dppz moiety, which is realized by threading of the Ru(phen)(2) complex part through the DNA duplex core. DNase I footprinting with four diastereomerically pure middle ([Ru(phen)(2)dppz](2+)-ODN).DNA duplexes furthermore showed that the tethered [Ru(phen)(2)dppz](2+)-linker chirality dictates the stereochemical accessibility of various phosphodiester moieties (around the intercalation site) toward the cleavage reaction by the enzyme. The diastereomerically pure ruthenium-modified duplexes, with the well-defined pi-stack, will be useful to explore stereochemistry-dependent energy- and electron-transfer chemistry to understand oxidative damage to the DNA double helix as well as the long-range energy- and electron-transfer processes with DNA as a reactant.

A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H.

The aim of this review is to draw a correlation between the structure of the DNA/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. The review is divided into the following parts: (1) mechanistic considerations, (2) target RNA folding-AON folding-RNase H assistance in AON/RNA hybrid formation, (3) carbohydrate modifications, (4) backbone modifications, (5) base modifications, (6) conjugated AONs, (7) importance of the tethered chromophore in AON for the AON/RNA hybrid interactions with the RNase H. The structural changes in the AON/RNA hybrid duplexes brought by different modifications of the sugar, backbone or base in the antisense strand, and the effect of these changes on the RNase H recognition of the modified substrates have been addressed. Only those AON modifications and the corresponding AON/RNA hybrids, which have been structurally characterized by spectroscopic means and functionally analyzed by their ability to elicit RNase H potency in comparison with the native counterpart have been presented here.

The identification of the A-type RNA helices in a 55mer RNA by selective incorporation of deuterium-labelled nucleotide residues (Uppsala NMR-window concept)

The 55-nt long RNA, modelling a three-way junction, with non-uniformly incorporated deuterated nucleotides has been synthesised in a pure form. The NMR-window part in this partially deuterated 55mer RNA consists of natural non-enriched nucleotide blocks at the three-way junction (shown in a square box in Fig. 2), whereas all other nucleotides of the rest of the molecule are partially deuterated (> 97 atom% 2H at C2', C3', C5', C5, and approximately 50 atom% 2H at C4'). The secondary structure of this 55mer RNA was determined by 2D 1H NOESY spectroscopy in D2O or in 10% D2O-H2O mixture. The use of deuterated building blocks in the specific region of the 55mer RNA allowed us to identify two distinct A-type RNA helices in a straightforward manner by observing connectivities of H1' with the basepaired imino and the aromatic H2 of all adenosine nucleotides as the first step for the determination of its tertiary structure in a cost- and time-effective manner without employing any 13C/15N labelling. These two decameric helices involve 40 nucleotides, for which all non-exchangeable H1', H6, H2, H8 and H5 protons (all 40 H1', all 40 H6 or H8 aromatics, all seven H2 of adenine nucleotide and all four non-deuterated H5 of cytosines) as well as all 16 exchangeable imino protons (with the exception of four terminal basepairs) and 16 amino protons of cytosines have been assigned. Since all aromatic-H2', H3' as well as H5'/5'' crosspeaks from partially deuterated residues have been eliminated from the NMR spectra, the observation of natural nucleotide residues in the NMR window part has essentially been simplified. It has been found that the crosspeaks from the natural nucleotides located at the three-way junction in the NMR-window part show different degrees of line-broadening, thereby indicating that the various nucleotide residues have very different mobilities with respect to themselves as well as compared to other nucleotides in the helices. The assignment of H2' and H3' in the NMR-window part has been made based on NOESY and DQF-COSY crosspeaks. It is noteworthy that, even in this preliminary study, it has been possible to identify 10 H2' out of total 14 and 9 H3' out of 14. The data show that expanded AU containing a tract of 55mer RNA does not self-organise into a tight third helix, as the two decameric A-type helices, across the three-way junction which is evident from the absence of any additional imino protons, except those that already have been assigned for the two decameric helices.