Sugar Pucker Research Articles

Synthesis and Photophysical Studies on N1-(2′-O,4′-C-Methyleneribofurano-nucleoside-3′-yl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles

A series of eight N1-(2′-O,4′-C-methylene-β-D-ribofuranonucleoside-3′-yl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles have been synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction of 3′-azido-3′-deoxy-2′-O,4′-C-methyleneuridine and 3′-azido-3′-deoxy-2′-O,4′-C-methylene-5-methyluridine with 7-propargyloxy coumarins in 82–88% yields. The synthesized coumarintriazolyl-bicyclonucleoside conjugates possess an extra bridge between 2′-O and 4′-C in the nucleoside moiety, which facilitates its pre-organization into N-type sugar puckering. This was confirmed by X-ray crystal structure studies on one of the conjugates, i.e. on N1-(3′-deoxy-2′-O,4′-C-methylene-5-methyluridin-3′-yl)-C4-(4-phenylcoumarin-7-oxymethyl)-1,2,3-triazole. Photophysical studies carried out on the synthesized compounds demonstrate that they possess useful level of fluorescence with Stokes shift of approximately 70 nm.

Crystal structure and sequential dehydration transitions of disodium guanosine 5'-monophosphate tetrahydrate.

Disodium guanosine 5'-monophosphate was reported previously to crystallize as both the tetrahydrate and the heptahydrate. We herein report a determination of the molecular and crystal structures of the title tetrahydrated salt, 2Na+·C10H12N5O8P2-·4H2O. It was found that the structure differs markedly from that of the heptahydrate, but greatly resembles that of disodium deoxyguanosine 5'-monophosphate tetrahydrate. The C2'-O2'H moiety of ribose is surrounded by hydrophilic moieties and is disordered over two sites. The sugar puckering mode is O4'-endo-C1'-exo at both sites and the conformation around the C4'-C5' bond is gauche-trans. Powder X-ray diffraction and thermal analyses revealed that the temperature-controlled transition from the tetrahydrate to the anhydride proceeded through three intermediate phases between 40 and 60 °C at 0% relative humidity. Large induction periods were observed.

Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Purine Nucleosides.

The chemical difference between DNA and RNA nucleosides is their 2'-hydrogen versus 2'-hydroxyl substituents. Modification of the ribosyl moiety at the 2'-position and 2'-O-methylation in particular, is common among natural post-transcriptional modifications of RNA. 2'-Modification may alter the electronic properties and hydrogen-bonding characteristics of the nucleoside and thus may lead to enhanced stabilization or malfunction. The structures and relative glycosidic bond stabilities of the protonated forms of the 2'-O-methylated purine nucleosides, 2'-O-methyladenosine (Adom) and 2'-O-methylguanosine (Guom), were examined using two complementary tandem mass spectrometry approaches, infrared multiple photon dissociation action spectroscopy and energy-resolved collision-induced dissociation. Theoretical calculations were also performed to predict the structures and relative stabilities of stable low-energy conformations of the protonated forms of the 2'-O-methylated purine nucleosides and their infrared spectra in the gas phase. Low-energy conformations highly parallel to those found for the protonated forms of the canonical DNA and RNA purine nucleosides are also found for the protonated 2'-O-methylated purine nucleosides. Importantly, the preferred site of protonation, nucleobase orientation, and sugar puckering are preserved among the DNA, RNA, and 2'-O-methylated variants of the protonated purine nucleosides. The 2'-substituent does however influence hydrogen-bond stabilization as the 2'-O-methyl and 2'-hydroxyl substituents enable a hydrogen-bonding interaction between the 2'- and 3'-substituents, whereas a 2'-hydrogen atom does not. Further, 2'-O-methylation reduces the number of stable low-energy hydrogen-bonded conformations possible and importantly inverts the preferred polarity of this interaction versus that of the RNA analogues. Trends in the CID50% values extracted from survival yield analyses of the 2'-O-methylated and canonical DNA and RNA forms of the protonated purine nucleosides are employed to elucidate their relative glycosidic bond stabilities. The glycosidic bond stability of Adom is found to exceed that of its DNA and RNA analogues. The glycosidic bond stability of Guom is also found to exceed that of its DNA analogue; however, this modification weakens this bond relative to its RNA counterpart. The glycosidic bond stability of the protonated purine nucleosides appears to be correlated with the hydrogen-bond stabilization of the sugar moiety.

DNA–RNA Hybrid Quadruplexes Reveal Interactions that Favor RNA Parallel Topologies

A DNA G-quadruplex adopting a (3+1)-hybrid structure was substituted at its 5'-tetrad by riboguanosine (rG) analogs. Incorporation of anti-favoring rG at appropriate syn-positions of the 5'-outer tetrad induced conformational rearrangements to yield a quadruplex featuring a 5'-tetrad with reversed polarity. A high-resolution structure of a disubstituted quadruplex variant as well as direct NMR experimental evidence reveals a non-conventional C-H⋅⋅⋅O hydrogen bond in a medium groove between the 2'-OH of an rG residue adopting a C2'-endo sugar pucker and H8 of a 3'-neighboring anti-G residue. In contrast, a C3'-endo sugar conformation for another guanine ribonucleotide prevents formation of a corresponding hydrogen bond but relocates its 2'-OH substituent from the quadruplex narrow groove into a medium groove. Both the formation of favorable CHO hydrogen bridges and unfavorable interactions of the 2'-hydroxyl group in a narrow groove will promote RNA folding into a parallel topology featuring all-anti core residues and four grooves of medium size.

Relative glycosidic bond stabilities of naturally occurring methylguanosines: 7-methylation is intrinsically activating.

The frequency and diversity of posttranscriptional modifications add an additional layer of chemical complexity beyond canonical nucleic acid sequence. Methylations are particularly frequently occurring and often highly conserved throughout the kingdoms of life. However, the intricate functions of these modified nucleic acid constituents are often not fully understood. Systematic foundational research that reduces systems to their minimum constituents may aid in unraveling the complexities of nucleic acid biochemistry. Here, we examine the relative intrinsic N-glycosidic bond stabilities of guanosine and five naturally occurring methylguanosines (O2'-, 1-, 7-, N2,N2-di-, and N2,N2,O2'-trimethylguanosine) probed by energy-resolved collision-induced dissociation tandem mass spectrometry and complemented with quantum chemical calculations. Apparent glycosidic bond stability is generally found to increase with increasing methyl substitution (canonical < mono- < di- < trimethylated). Many biochemical transformations, including base excision repair mechanisms, involve protonation and/or noncovalent interactions to increase nucleobase leaving-group ability. The protonated gas-phase methylguanosines require less activation energy for glycosidic bond cleavage than their sodium cationized forms. However, methylation at the N7 position intrinsically weakens the glycosidic bond of 7-methylguanosine more significantly than subsequent cationization, and thus 7-methylguanosine is suggested to be under perpetually activated conditions. N7 methylation also alters the nucleoside geometric preferences relative to the other systems, including the nucleobase orientation in the neutral form, sugar puckering in the protonated form, and the preferred protonation and sodium cation binding sites. All of the methylated guanosines examined here are predicted to have proton affinities and gas-phase basicities that exceed that of canonical guanosine. Additionally, the proton affinity and gas-phase basicity trends exhibit a roughly inverse correlation with the apparent glycosidic bond stabilities.

Effect of osmolytes of different type on DNA behavior in aqueous solution. Experimental and theoretical studies

Osmolytes, the small organic molecules accumulated in cells under environmental stress, can modulate the stability of biopolymers such as proteins and DNA. In spite of many years of research, there is no established molecular mechanism of the influence of osmolytes on DNA structure. Here, we used two model osmolytes that denature (urea) or stabilize (trimethylglycine, TMG) proteins to study their effect on DNA in aqueous solutions using a combination of spectroscopic, calorimetric and simulation methods. We found that both urea and TMG dehydrate DNA and shift the conformational equilibrium from B- to A-DNA form, despite different solvation properties. The isolated vibrational spectra of osmolytes and analysis of preferential accumulation indicate a lack of direct specific interaction with DNA in ternary solutions. Besides the influence of TMG and urea on DNA hydration, we studied and analyzed the orientational and spatial distribution of osmolytes around DNA. Analysis of hydrogen bonds and theoretical calculations of interactions with native and denatured DNA show strong negative interactions with bases of dissociated DNA: while TMG preferentially interacts with guanine, urea interacts equally strongly with all bases. This high affinity of both TMG and urea towards DNA bases appears to be the cause of their denaturing properties.

Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile

Different forms of viruses that infect archaea inhabiting extreme environments continue to be discovered at a surprising rate, suggesting that the current sampling of these viruses is sparse. We describe here Sulfolobus filamentous virus 1 (SFV1), a membrane-enveloped virus infecting Sulfolobus shibatae. The virus encodes two major coat proteins which display no apparent sequence similarity with each other or with any other proteins in databases. We have used cryo-electron microscopy at 3.7 Å resolution to show that these two proteins form a nearly symmetrical heterodimer, which wraps around A-form DNA, similar to what has been shown for SIRV2 and AFV1, two other archaeal filamentous viruses. The thin (∼ 20 Å) membrane of SFV1 is mainly archaeol, a lipid species that accounts for only 1% of the host lipids. Our results show how relatively conserved structural features can be maintained across evolution by both proteins and lipids that have diverged considerably.

Modified Quadrupole Ion Trap Mass Spectrometer for Infrared Ion Spectroscopy: Application to Protonated Thiated Uridines.

Modifications to a Paul-type quadrupole ion trap mass spectrometer providing optical access to the trapped ion cloud as well as hardware and software for coupling to a table-top IR optical parametric oscillator laser (OPO) are detailed. Critical experimental parameters for infrared multiple photon dissociation (IRMPD) on this instrument are characterized. IRMPD action spectra, collected in the hydrogen-stretching region with this instrument, complemented by spectra in the IR fingerprint region acquired at the FELIX facility, are employed to characterize the structures of the protonated forms of 2-thiouridine, [s2Urd+H]+, and 4-thiouridine, [s4Urd+H]+. The measured spectra are compared with predicted linear IR spectra calculated at the B3LYP/6-311+G(d,p) level of theory to determine the conformers populated in the experiments. This comparison indicates that thiation at the 2- or 4-positions shifts the protonation preference between the 2,4-H tautomer and 4-protonation in opposite directions versus canonical uridine, which displays a roughly equal preference for the 2,4-H tautomer and O4 protonation. As found for canonical uridine, protonation leads to a mixture of conformers exhibiting C2'-endo and C3'-endo sugar puckering with an anti nucleobase orientation being populated for both 2- and 4-thiated uridine. Graphical Abstract ᅟ.

Adjusting the Structure of 2'-Modified Nucleosides and Oligonucleotides via C4'-α-F or C4'-α-OMe Substitution: Synthesis and Conformational Analysis.

We report the first syntheses of three nucleoside analogues, namely, 2',4'-diOMe-rU, 2'-OMe,4'-F-rU, and 2'-F,4'-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4'-α-position of a 4',5'-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2',4'-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4'-α-F/OMe substituents reinforce the C3'-endo ( north) conformation of 2'-OMe-rU. Furthermore, the predominant C2'-endo ( south/ east) conformation of 2'-F-araU switches to C3'-endo upon introduction of these substituents at C4'. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4'-substituents can modulate the binding affinity of the parent 2'-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics.

Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4′-C-aminoalkyl-2′-O-methyluridine adopts an C2′-endo sugar puckering in dsRNA. The 4′-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.

Towards Electronic Detection of DNA Conformational Transition

The conformation of DNA plays an important role in its chemical and biological function as well as for its potential applications in nanotechnology1. Different conformations of DNA exhibit distinct values for the conductance2. Such a distinct conductance can also be used to detect the conformation. DNA mainly adopts A and B conformations at lower and higher hydration levels, respectively1,2. Experimental and theoretical studies suggest that A to B transformation is smooth and continuous1, whereas B to A transition is sequence specific, cooperative, and reversible3,4. To the best of our knowledge, there is no experimental method that can precisely evaluate the electronic structure of DNA during its conformation transition. A recent theoretical study discusses the change in electronic properties during the transformation1. A recent work found an unexpected result that A-DNA conducts better than B-DNA. To provide insight into the time evolution of the electronic properties during change in conformation, we present a computational investigation of the electrical conductance of 3’-CCCGCGCCC-5’ DNA during various time step of the MD trajectory as the molecule undergoes A to B transition. The initial structure of A-DNA is built using nucleic acid builder (NAB) in AMBER15. For DNA, the parmbsc1 force field is used and SPC/E force field is used for water and ions. The negative charge on DNA backbone is neutralized with sodium (Na) ions. Additionally, Na+ and Cl- ions are added to achieve 150mM salt concentration. For A to B DNA transformation, A-DNA is placed in water. Further minimization and equilibration is performed according to ABC Minimization5. 50 ns molecular dynamics (MD) trajectory is generated with particle mesh Ewald (PME) algorithm and periodic boundary condition (PBC) under constant pressure (isobaric-isothermal ensemble). The transformed B-DNA at the end of 50ns simulation is then placed in 85%ethanol+15%water with 150mM of salt concentration. Packmol is used to model the 85%ethanol+15%water solvent. The temperature is increased to 325K and the simulation is continued for another 48ns. The snapshots during A to B transformations (at 1ps, 50 ps, 100ps, 125ps, 150ps, 200ps and 50000ps, respectively) are considered for electronic structure calculations, where the DNA encounters major structural changes (also done for B to A transformation). Density Functional Theory (DFT) calculations are performed on these structures using GAUSSIAN 09, with the B3LYP functional and 6-31G(d,p) basis set. The electrical conductance of DNA is determined using Landauer formalism with Non-equilibrium Green's function approach2. Figure 1 (a) shows the sugar pucker in the range of 0o-36o (representing A-DNA) in the initial phase of simulation that increases step by step to 140o-180o (representing B-DNA) and remains in that range till 50ns. We also find that the rise per base pairs increases from ~2.6Å to ~3.4 Å and twist increases from 30-34 Å to 35-38 Å, which confirm the transformation. We could reverse this transformation when the B-DNA is placed in 85%ethanol+15%water at 325K (results not presented here). A-DNA at 1ps has HOMO at -5.04eV and LUMO at -1.04eV. During the transformation, mean value of HOMO energy is -5.06eV and that of LUMO is -1.21eV, indicating significant change in the LUMO energy (~20%). We next look at the charge transport during the transformation, i.e. for the frames at 1 ps, 50 ps, 100 ps, 125 ps, 150ps, 200ps and 50000ps. Overall conductance is seen to decrease (increase) with the A(B) to B(A) transformation (Figure 1 (b) for A to B transformation). It is to be noted that this is the same DNA sequence investigated experimentally2 for which the A-conformation exhibits higher conductance than B-conformation. Our simulations clearly verify these experimental findings and provide further information on the evolution of conductance with time. The fluctuations in between could be related to the spatial configurations of counterions, which can alter the conductance of DNA6. References 1 H. Wang, T.E. Cheatham, P.M. Gannett, and J.P. Lewis, Soft Matter 5, 685 (2009). 2 J.M. Artés, Y. Li, J. Qi, M.P. Anantram, and J. Hihath, Nat. Commun. 6, (2015). 3 J.T. Waters, X.-J. Lu, R. Galindo-Murillo, J.C. Gumbart, H.D. Kim, T.E. Cheatham, and S.C. Harvey, J. Phys. Chem. B 120, 8449 (2016). 4 M. Kulkarni and A. Mukherjee, Prog. Biophys. Mol. Biol. 128, 63 (2017). 5 M. Pasi, J.H. Maddocks, D. Beveridge, T.C. Bishop, D.A. Case, I.I.I.T. Cheatham, P.D. Dans, B. Jayaram, F. Lankas, C. Laughton, J. Mitchell, R. Osman, M. Orozco, A. Pérez, D. Petkevičiūtė, N. Spackova, J. Sponer, K. Zakrzewska, and R. Lavery, Nucleic Acids Res. 42, 12272 (2014). 6 R.N. Barnett, C.L. Cleveland, A. Joy, U. Landman, and G.B. Schuster, Science (80-. ). 294, 567 (2001). Figure 1

The crystallization process, base pair and hydrogen-bond network of guanosine derivatives-based hydrogel

In the past few decades, supramolecular gels self-assembled from low-molecular-weight gelators are attractive for various applications including regenerative medicine and drug delivery. Although guanosine-based hydrogel have a long history, a major limitation was their poor lifetime stability on account of crystallization within hours at room temperature. However, the crystallization process of guanosine-based hydrogel has not been explored before. In this work, the crystallization process of 2′-deoxy-2′-fluoroguanosine (FGd) hydrogel has been studied by single-crystal X-ray analysis and scanning electron microscope (SEM). The results indicated that the crystallization from the FGd hydrogel is divided into two phases: small flake-like and bigger piece-like crystals. Compared the flake-like with piece-like crystal, we found that they display similar torsion angle, sugar puckering, base pair and hydrogen bond patterns with subtle distinctions for the sugar-sugar and base-sugar interactions. This study not only enriches the structural varieties of guanosine derivatives, but also provides specific information to further understand how to design new guanosine-based gelators in the future.

Atomic structures of excited state A-T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations.

NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3' and C4' spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.

Characterization of the secondary structure and protein binding affinity of an intramolecular DNA four‐way junction.

Oligodeoxynucleotides (ODN) offer great promise as antigene and antisense reagents due to their high binding specificity, straight‐forward synthesis and low toxicity. Research shows that ODNs can also target proteins such as the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a redox sensitive damage associated molecular pattern (DAMP) molecule. And several studies link deleterious HMGB1 inflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 is gathering a great deal of attention as a biomarker and therapeutic target. We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. In order to enhance the stability of 4WJs for ex vivo studies, the helical termini of a DNA junction are modified to generate the intramolecular construct, i‐J1. The secondary structure, thermostability and protein binding affinity of i‐J1 are investigated vs. a control 4WJ (J1) and the intramolecular 4WJ, J4. Non‐denaturing gel electrophoresis assays show that i‐J1 possesses a global structural conformation that is analogous to the control 4WJ. Circular dichroism (CD) studies show that the secondary structure of i‐J1 is composed of B‐ and A‐form DNA helices. Moreover, the melting temperature (Tm) of i‐J1 is significantly higher than J1. The Tm of i‐J1 is ~ 30°C higher than J1. Finally, electrophoretic mobility shift assays (EMSAs) show that the HMG protein, HMGB1b, binds i‐J1 with nearly identical affinity to J1. The data clearly shows that the junction lattice of a DNA four‐way junction can be altered to enhance conformational stability and retain protein binding affinity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The C2′- and C3′-endo equilibrium for AMP molecules bound in the cystathionine-beta-synthase domain

The equilibrium between C2′- and C3′-endo conformations of nucleotides in solution, as well as their polymers DNA and RNA, has been well studied in previous work. However, this equilibrium of nucleotides in their binding state remains unclear. We observed two AMP molecules, in C3′- and C2′-endo conformations respectively, simultaneously bound to a cystathionine-beta-synthase (CBS) domain dimer of the magnesium and cobalt efflux protein CorC in the crystallographic study. The C2′-endo AMP molecule assumes the higher sugar pucker energy and one more hydrogen bond with the protein than the C3′-endo molecule does. The balance between the high sugar pucker energy and the low binding energy suggests an equilibrium or switch between C2′- and C3′-endo conformations of the bound nucleotides. Our work challenge the previous hypothesis that the ribose of the bound nucleotides would be locked in a fixed conformation.

AMOEBA Polarizable Atomic Multipole Force Field for Nucleic Acids.

The AMOEBA polarizable atomic multipole force field for nucleic acids is presented. Valence and electrostatic parameters were determined from high-level quantum mechanical data, including structures, conformational energy, and electrostatic potentials, of nucleotide model compounds. Previously derived parameters for the phosphate group and nucleobases were incorporated. A total of over 35 μs of condensed-phase molecular dynamics simulations of DNA and RNA molecules in aqueous solution and crystal lattice were performed to validate and refine the force field. The solution and/or crystal structures of DNA B-form duplexes, RNA duplexes, and hairpins were captured with an average root-mean-squared deviation from NMR structures below or around 2.0 Å. Structural details, such as base pairing and stacking, sugar puckering, backbone and χ-torsion angles, groove geometries, and crystal packing interfaces, agreed well with NMR and/or X-ray. The interconversion between A- and B-form DNAs was observed in ethanol-water mixtures at 328 K. Crystal lattices of B- and Z-form DNA and A-form RNA were examined with simulations. For the RNA tetraloop, single strand tetramers, and HIV TAR with 29 residues, the simulated conformational states, 3 J-coupling, nuclear Overhauser effect, and residual dipolar coupling data were compared with NMR results. Starting from a totally unstacked/unfolding state, the rCAAU tetranucleotide was folded into A-form-like structures during ∼1 μs molecular dynamics simulations.

Molecular Dynamics Simulations Reveal an Interplay between SHAPE Reagent Binding and RNA Flexibility.

The function of RNA molecules usually depends on their overall fold and on the presence of specific structural motifs. Chemical probing methods are routinely used in combination with nearest-neighbor models to determine RNA secondary structure. Among the available methods, SHAPE is relevant due to its capability to probe all RNA nucleotides and the possibility to be used in vivo. However, the structural determinants for SHAPE reactivity and its mechanism of reaction are still unclear. Here molecular dynamics simulations and enhanced sampling techniques are used to predict the accessibility of nucleotide analogs and larger RNA structural motifs to SHAPE reagents. We show that local RNA reconformations are crucial in allowing reagents to reach the 2′-OH group of a particular nucleotide and that sugar pucker is a major structural factor influencing SHAPE reactivity.

Studies on sugar puckering and glycosidic stabilities of 3'-amino-5'-carboxymethyl-3',5'-dideoxy nucleoside mimics.

The synthesis of nucleoside amino acid monomers and dimers has been carried out to evaluate and characterize the impact of the neutral amide backbone on key attributes like puckering of the sugar rings and glycosidic bond strengths of these analogs. The conformational analysis suggests that amide-linked nucleotides have a high predilection towards N-type conformers. The glycosidic bond strength was found to be slightly weaker compared to ribonucleosides under acidic conditions at high temperatures. The results will be helpful to explore in future the development of fully amide-linked oligonucleotides for therapeutic purposes.

A- to B-DNA Transition in AMBER Force Fields and Its Coupling to Sugar Pucker.

The A/B transition is a basic element of DNA conformational change. Because of its involvement in the sensing of the ionic conditions by DNA and in specific protein-DNA interactions, this transition is important for biological functions of DNA. Therefore, accurate modeling of the A/B equilibrium by means of empirical force fields is of utmost interest. In this work, we examine the A/B equilibrium in three AMBER force fields, including the recent bsc1 and OL15 modifications, using much longer MD simulations than attempted before. Special attention is paid to the coupling of the A/B equilibrium with the south/north (S/N) transition of the sugar pucker. We found that none of the tested force fields provided a satisfactory description of the A/B equilibrium because the B-form was predicted to be much too stable and the A-form was predicted to be almost absent even in concentrated trifluoroethanol solutions. Based on comparison with NMR data for duplexes and single nucleosides, we hypothesize that this problem arose from the incorrect description of the S/N equilibrium of sugar pucker, where the south conformation is much too stable, thus stabilizing the B-form. Because neither the A/B equilibrium in duplexes nor the S/N equilibrium in nucleosides was described accurately, further refinements of the AMBER DNA force fields are needed.

A nucleobase-centered coarse-grained representation for structure prediction of RNA motifs.

We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the n}{}mathcal {E}SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base–phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation.