45 research outputs found
Sequence-specific Solution Structures of the Four Isosequential Pairs of Single-stranded DNAs and RNAs
The role of the sequence-context in the self-organization of four single-stranded (ss) isosequential pairs of DNAs (1 – 4) and RNAs (5 – 8), [d/r-(5'C^1^A^2^X^3^G^4^Y^5^A^6^C^7^): X^3^ = A or C, Y^5^ = A or C; sequence variations: 2^2^ = 4], has been elucidated by NMR-constrained Molecular Dynamics (MD) simulations (2 ns). Following sequence-specific observations have been made from the solution NMR and the NMR constrained MD simulation study: (i) Analysis of the NOESY footprints, mainly (H8/H6)~n~ to (H1' and H3')~n-1~ contacts, of ssDNAs (1 - 4) and ssRNAs (5 – 8) in the aqueous medium have shown that all ssDNAs (1 - 4) and ssRNAs (5 - 8) adopt right handed stacked helical structures in the NMR time scale. (ii) Intra-residual cross-peak intensities for the H(8/6)~n-~ H(1'/2'/2''/H3')~n~ contacts in ssDNAs and ssRNAs are stronger at the 3'-ends in comparison with those at the 5'-ends, suggesting that the dynamics of the nucleobases at the 3'-end are more restricted, whereas those at the 5'-end are more flexible. (iii) This relative NMR found mobility is consistent with the final RMSd calculations of the final NMR-MD structures of ssDNAs and ssRNAs. They show that the 5'-end nucleobases have higher RMSd values compared to those at the 3'-end, except for the sequence d/r(5'C^1^A^2^A^3^G^4^A^5^A^6^C^7^). (iv) Relative nOe intensities of inter-residual H(8/6)~n~ - H(1')~n-1~ and H(8/6)~n~ - H(3')~n-1~ contacts, as well as NMR observed fluctuations in the sugar conformations, for ssDNAs (1 – 4) and ssRNAs (5 – 8) show that no ssDNA or ssRNA adopts either a typical B-type DNA or A-type RNA form. (v) In the final NMR-MD structures all the [H8/6N~(n)~ -- H1'N~(n-1)~/ H3'N~(n-1)~, N = A, G, C] distances in different isosequential pairs of ssDNA (1 – 4) and ssRNA (5 – 8) change depending upon the sequence context of the single-stranded nucleic acids. Both in the deoxy and ribo series, it is the purine-rich sequences [d/r-(5'C^1^A^2^A^3^G^4^A^5^A^6^C^7^) which form the most stable self-organized right-handed helical structures because of the favorable purine-purine stacking interactions. (vi) Stacking pattern at each of the dinucleotide steps show that the base-base nearest neighbor stacking interactions depend solely upon the sequence contexts of the respective ssDNAs (1 – 4) and ssRNAs (5 – 8). See pages 47 – 145 for Supplementary Information for detailed spectroscopic data
A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low
Synthesis and structure of azole-fused indeno[2,1-c]quinolines and their anti-mycobacterial properties †
Prompted by our discovery of a new class of conformationally-locked indeno[2,1-c]quinolines as anti-mycobacterials, compounds 2a and 3a
A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity
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Electrostatic cross-modulation of the pseudoaromatic character in single-stranded RNA by nearest-neighbor interactions
The generation of a single anionic or cationic center at an alkaline or acidic pH in a given molecule presents a unique opportunity to examine the electrostatic make-up of these molecules both at the neutral or ionic state. The generation of a single cationic center in the phenyl-nicotinamide system provided new straightforward evidence showing that the charge density of the electron-deficient pyridinium was actually enhanced by the donation of the charge from the electron-rich phenyl group (i.e., the pyridinyl became more basic by ca. 0.5 pKa unit compared to an analogous system where phenyl was absent) owing to the electrostatic interactions between these two moieties. On the other hand, the generation of the 5'-guanylate ion in the hexameric single-strand (ss) RNA [5'-GAAAAC-3'], in comparison with the constituent trimeric, tetrameric, and pentameric-ssRNAs, has unequivocally shown how far the electrostatic cross-talk (as an interplay of Coulombic attractive or repulsive forces) in this electronically coupled system propagates through the intervening pAp nucleotide steps until the terminal pC-3' residue in comparison with the neutral counterpart. The footprint of the propagation of this electrostatic cross-talk among the neighboring nucleobases is evident by measurement of pKas from the marker protons of ionization point (i.e., of G) as well as from the neighboring marker protons (i.e., of A or C) in the vicinity, as well as from the change of the chemical environment (i.e., chemical shifts) around their aromatic marker protons (δH2, δH8, δH5, and δH6) owing to a change of the stacking →← destacking equilibrium as a function of pH
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The hydrogen bonding and hydration of 2′-OH in adenosine and adenosine 3′-ethyl phosphate
The 2‘-OH group has major structural implications in the recognition, processing, and catalytic properties of RNA. We report here intra- and intermolecular H-bonding of 2‘-OH in adenosine 3‘-ethyl phosphate (1), 3‘-deoxyadenosine (2), and adenosine (3) by both temperature- and concentration-dependent NMR studies, as well as by detailed endo (3JH,H) and exocyclic (3JH,OH) coupling constant analyses. We have also examined the nature of hydration and exchange processes of 2‘-OH with water by a combination of NOESY and ROESY experiments in DMSO-d6 containing 2 mol % HOD. The NMR-constrained molecular modeling (by molecular mechanics as well as by ab initio methods both in the gas and solution phase) has been used to characterize the energy minima among the four alternative dihedrals possible from the solution of the Karplus equation for 3JH2‘,OH and 3JH3‘,OH to delineate the preferred orientation of 2‘-O-H proton in 1 and 2 as well as for 2‘/3‘-O-H protons in 3. The NMR line shape analysis of 2‘-OH gave the Δ of 7.5 kJ mol-1 for 1 and 8.4 kJ mol-1 for 3; similar analyses of the methylene protons of 3‘-ethyl phosphate moiety in 1 also gave comparable Δ of 7.3 kJ mol-1. The donor nature of the 2‘-OH in the intramolecular H-bonding in 3 is evident from its relatively reduced flexibility [−TΔS⧧]2‘-OH = −17.9(±0.5) kJ mol-1] because of the loss of conformational freedom owing to the intramolecular 2‘O-H···O3‘ H-bonding, compared to the acceptor 3‘-OH in 3 [−TΔS⧧]3‘-OH = −19.8 (± 0.6) kJ mol-1] at 298 K. The presence of intramolecular 2‘-OH···O3‘ H-bonding in 3 is also corroborated by the existence of weak long-range 4JH2‘,OH3‘ in 3 (i.e., W conformation of H2‘−C2‘−C3‘−O3‘−H) as well as by 3JH,OH dependent orientation of the 2‘- and 3‘-OH groups. The ROESY spectra for 1 and 3 at 308 K, in DMSO-d6, show a clear positive ROE contact of both 2‘- and 3‘-OH with water. The presence of a hydrophilic 3‘-phosphate group in 1 causes a much higher water activity in the vicinity of its 2‘-OH, which in turn causes the 2‘-OH to exchange faster, culminating in a shorter exchange lifetime (τ) for 2‘-OH proton with HOD in 1 (τ2‘-OH: 489 ms) compared to that in 3 (τ2‘-OH: 6897 ms). The activation energy (Ea) of the exchange with the bound-water for 2‘- and 3‘-OH in 3 (48.3 and 45.0 kJ mol-1, respectively) is higher compared to that of 2‘-OH in 1 (31.9 kJ mol-1), thereby showing that the kinetic availability of hydrated 2‘-OH in 1 for any inter- and intramolecular interactions, in general, is owing to the vicinal 3‘-phosphate residue. It also suggests that 2‘-OH in native RNA can mediate other inter- or intramolecular interactions only in competition with the bound-water, depending upon the specific chemical nature and spatial orientation of other functions with potential for hydrogen bonding in the neighborhood. This availability of the bound water around 2‘-OH in RNA would, however, be dictated by whether the vicinal phosphate is exposed to the bulk water or not. This implies that relatively poor hydration around a specific 2‘-OH across a polyribonucleotide chain, owing to some hydrophobic microenvironmental pocket around that hydroxyl, may make it more accessible to interact with other donor or acceptor functions for H-bonding interactions, which might then cause the RNA to fold in a specific manner generating a new motif leading to specific recognition and function. Alternatively, a differential hydration of a specific 2‘-OH may modulate its nucleophilicity to undergo stereospecific transesterification reaction as encountered in ubiquitous splicing of pre-mRNA to processed RNA or RNA catalysis, in general
The Strength of the Anomeric Effect in Adenosine, Guanosine, and in Their 2′-Deoxy Counterparts is Medium-Dependent
In nucleosides, the anomeric effect (AE) (i.e. stereoelectronic n(O4′)fσ* C1′-N9 interactions) places the aglycon in the pseudoaxial orientation in the N-type conformation (2′-exo-3′-endo), whereas the inherent steric effect of the nucleobase opposes the AE by its tendency to take up pseudoequatorial orientation in the S-type conformation (2′-endo-3′-exo). This means that the actual energetic contribution of the AE of an N-or a C-aglycon in a nucleoside can be determined by subtracting the steric effect of the N-or C-aglycon from the total effect of the aglycon on the drive of N a S pseudorotational equilibrium. The ∆G°of N a S pseudorotational equilibrium among a set of various neutral C-and N-nucleosides showed that the relatively most thermodynamically stabilized S-type conformer is found in 9-deazaadenosine in which 9-deazaadenin-9-yl at C1′ takes up the relatively most favored pseudoequatorial orientation between pH 8.8-12.0 (∆H°) -14.2 kJ/mol) as a result of the exclusive steric control for the drive (∆H°) of N a S pseudorotational equilibrium. 9-Deazaadenin-9-yl at C1′ therefore serves as the best reference point for subtraction of the steric effect of the adenin-9-yl or guanin-9-yl in adenosine (A), guanosine (G), and in their 2′-deoxy counterparts (dA and dG). Since the electronic character of adenin-9-yl or guanin-9-yl changes from the neutral to the protonated (or deprotonated in case of guanin-9-yl) state as the pH of the medium changes (refs 1p, 1s), the work reported here shows for the first time that the intrinsic AE of A, G, dA, and dG are indeed pD-dependent. The tunable strength of the AE can vary from 23.4 to 17.7 kJ/mol in A from pD 1.2 to 7.0, 37.5 to 15.6 kJ/mol in G from pD 0.6 to 11.6, 18.0 to 14.8 kJ/mol in dA from pD 0.9 to 7.0, 20.7 to 13.8 kJ/mol in dG from pD 0.9 to 11.6