Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24–29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3′-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.Instituto de Salud Carlos III [Miguel Servet Program, CP13/00211, 205024141 to M.T.]; Spanish MINECO [BIO2012–32869 and BIO2015-64802-R toM.O.]; AGAUR (toM.O.); ERCCouncil (SimDNA, grant 291433, to M.O.). M.O. is an ICREA Academia fellow. Funding for open access charge: ERC Council [grant 291433 (simDNA)].Peer ReviewedPostprint (published version

How accurate are accurate force-fields for B-DNA?

Last generation of force-fields are raising expectations on the quality of molecular dynamics (MD) simulations of DNA, as well as to the belief that theoretical models can substitute experimental ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing 'experimental models', which in turn, have an unclear accuracy to represent DNA conformation in solution. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, determined herein by means of 1H nuclear magnetic resonance (NMR). The study allowed us to check directly for experimental NMR observables on duplexes previously not solved, and also to assess the reliability of 'experimental structures'. We observed that technical details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theoretical simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details

The structural impact of DNA mismatches

ABSTRACT The structure and dynamics of all the transversion and transition mismatches in three different DNA environments have been characterized by molecular dynamics simulations and NMR spectroscopy. We found that the presence of mismatches produced significant local structural alterations, especially in the case of purine transversions. Mismatched pairs often show promiscuous hydrogen bonding patterns, which interchange among each other in the nanosecond time scale. This therefore defines flexible base pairs, where breathing is frequent, and where distortions in helical parameters are strong, resulting in significant alterations in groove dimension. Even if the DNA structure is plastic enough to absorb the structural impact of the mismatch, local structural changes can be propagated far from the mismatch site, following the expected through-backbone and a previously unknown through-space mechanism. The structural changes related to the presence of mismatches help to understand the different susceptibility of mismatches to the action of repairing proteins

Small Details Matter: The 2'-Hydroxyl as a Conformational Switch in RNA

While DNA is mostly a primary carrier of genetic information and displays a regular duplex structure, RNA can form very complicated and conserved 3D structures displaying a large variety of functions, such as being an intermediary carrier of the genetic information, translating such information into the protein machinery of the cell, or even acting as a chemical catalyst. At the base of such functional diversity is the subtle balance between different backbone, nucleobase, and ribose conformations, finely regulated by the combination of hydrogen bonds and stacking interactions. Although an apparently simple chemical modification, the presence of the 2′OH in RNA has a profound effect in the ribonucleotide conformational balance, adding an extra layer of complexity to the interactions network in RNA. In the present work, we have combined database analysis with extensive molecular dynamics, quantum mechanics, and hybrid QM/MM simulations to provide direct evidence on the dramatic impact of the 2′OH conformation on sugar puckering. Calculations provide evidence that proteins can modulate the 2′OH conformation to drive sugar repuckering, leading then to the formation of bioactive conformations. In summary, the 2′OH group seems to be a primary molecular switch contributing to specific protein–RNA recognition

Long-timescale dynamics of the Drew-Dickerson dodecamer

We present a systematic study of the long-timescale dynamics of the Drew-Dickerson dodecamer (DDD: d(CGCGAATTGCGC)2) a prototypical B-DNA duplex. Using our newly parameterized PARMBSC1 force field, we describe the conformational landscape of DDD in a variety of ionic environments from minimal salt to 2 M Na(+)Cl(-) or K(+)Cl(-) The sensitivity of the simulations to the use of different solvent and ion models is analyzed in detail using multi-microsecond simulations. Finally, an extended (10 μs) simulation is used to characterize slow and infrequent conformational changes in DDD, leading to the identification of previously uncharacterized conformational states of this duplex which can explain biologically relevant conformational transitions. With a total of more than 43 μs of unrestrained molecular dynamics simulation, this study is the most extensive investigation of the dynamics of the most prototypical DNA duplex

Sciatic lateral popliteal block with clonidine alone or clonidine plus 0.2% ropivacaine: effect on the intra-and postoperative analgesia for lower extremity surgery in children: a randomized prospective controlled study

<p>Abstract</p> <p>Background</p> <p>The effect of adding clonidine to local anesthetics for nerve or plexus blocks remains unclear. Most of the studies in adults have demonstrated the positive effects of clonidine on intra- and postoperative analgesia when used as an adjunctive agent or in some cases as a single to regional techniques. In the pediatric population, there are only few trials involving clonidine as an adjunct to regional anesthesia, and the analgesic benefits are not definite in this group of patients. The evidence concerning perineural administration of clonidine is so far inconclusive in children, as different types and volume of local anesthetic agents have been used in these studies. Moreover, the efficacy of regional anesthesia is largely affected by the operator's technique, accuracy and severity of operation.</p> <p>Methods</p> <p>The use of clonidine alone or combined with 0.2% ropivacaine for effective analgesia after mild to moderate painful foot surgery was assessed in 66 children, after combined sciatic lateral popliteal block (SLPB) plus femoral block. The patients were randomly assigned into three groups to receive placebo, clonidine, and clonidine plus ropivacaine. Time to first analgesic request in the groups was analyzed by using Kaplan-Meier and the log-rank test (mean time, median time, 95% CI).</p> <p>Results</p> <p>In our study, clonidine administered alone in the SLPB seems promising, maintaining intraoperatively the hemodynamic parameters SAP, DAP, HR to the lower normal values so that no patient needed nalbuphine under 0.6 MAC sevoflurane anesthesia, and postoperatively without analgesic request for a median time of 6 hours. In addition, clonidine administered as adjuvant enhances ropivacaine's analgesic effect for the first postoperative day in the majority of children (p = 0.001). Clonidine and clonidine plus ropivacaine groups also didn’t demonstrate PONV, motor blockade, and moreover, the parents of children expressed their satisfaction with the excellent perioperative management of their children, with satisfaction score 9.74 ± 0.45 and 9.73 ± 0.70 respectively. On the contrary all the patients in the control group required rescue nalbuphine in the recovery room, and postoperatively, along with high incidence of PONV, and the parents of children reported a low satisfaction score (7.50 ± 0.70).</p> <p>Conclusions</p> <p>Clonidine appears promising more as an adjuvant in 0.2% ropivacaine and less than alone in the SLPB plus femoral block in children undergoing mild to moderate painful foot surgery, with no side effects.</p> <p>Trial registration</p> <p>ClinicalTrials.gov, <a href="http://www.controlled-trials.com/ISRCTN90832436">ISRCTN90832436</a>, (ref: CCT-NAPN-20886).</p

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo

Parmbsc1: A refined force-field for DNA simulations

We present parmbsc1, a force field for DNA atomistic simulation, which has been parameterized from high-level quantum mechanical data and tested for nearly 100 systems (representing a total simulation time of ∼140 μs) covering most of DNA structural space. Parmbsc1 provides high-quality results in diverse systems. Parameters and trajectories are available at http://mmb.irbbarcelona.org/ParmBSC1/

Parmbsc1: Parameterization and Validation of a new State-of-the-art Force Field for DNA Simulations

[eng] Classical force fields are the core of classical simulations, particularly of molecular dynamics (MD), a technique that is changing our view on the structure, flexibility and function of biological macromolecules. Originated from the pioneering work of Lifson’s group in the sixties, force fields have been in continuous evolution, improving in each generation the accuracy in the representation of proteins and nucleic acid. Force field development is tightly connected to the refinement of simulation procedures and to the extension of simulation time scales. Thus, as simulation time passed the microsecond barrier, MD simulations have revealed the existence of some errors in the default force field for DNA simulations, parmbsc0 (developed in the group). The goal of this thesis is to address these problems by a reparameterization of AMBER force field that aims to represent a wide range of DNA structures under physiological and non-physiological conditions. Keeping α/γ parmbsc0 corrections and parm99 non-bonded parameters, we systematically reparameterized sugar puckering, ε, ζ and χ torsions using high level QM calculations both in gas phase and solution. The refined force field has been tested for more than 3 years to an unprecedented level of detail, considering a large variety of DNAs, and analyzing structural, mechanical and dynamical properties of the DNAs resulting from the corresponding MD simulations. The refined force field parameters have been also subjected for more than 1 year of β-testing by different groups, finding to our knowledge no major drawbacks. In the world of RNA simulations, despite the recent efforts to improve the description of RNA in MD simulations, RNA force fields are still far in accuracy from those of DNA. A probable cause could be the incomplete understanding of the mechanism of 2’-OH orientation, which in big extent determines the RNA conformation and most probably serves as the molecular switch. THESIS ORGANIZATION This thesis is compiled of five publications (or in the process of publication) works; first three consider DNA force field development and following validation and benchmark while the last two are focused on RNA efforts. For better understanding of this work Chapter 1 introduces the central concepts related to nucleic acids, their structures and ways to study them. Chapter 2 goes into more details of the methodology employed here, briefly explaining basic QM formalism and MD simulations with an emphasis on force fields. Chapter 3 is a small handbook of methods employed in the analysis in this work. All together first three chapters should provide a solid ground to better understand the details and the relevance of the five publications in the following two chapters. Chapter 4 is based on the development of new force field, called parmbsc1, its further testing on the Drew-Dickerson sequence and benchmarking. Chapter 5 focuses on efforts to understand the mechanism of complexity of RNA structures studying 2’-OH rotation, and computational design of a new RNA dumbbell structure. A summary of the major results and a general discussion that reflects on the five projects and future work are presented in Chapter 6, with the main conclusions at the end of this work

Použití moderních výpočetních metod pro simulace molekulárních spekter

Přesné výpočty vibračních energií a vibračních spekter molekul vyžadují započítání anharmonických sil. Ve standardním výpočetním protokolu, jsou velké vibrační matice Hamiltoniánu diagonalizovány a spektrální intenzity počítány pro jednotlivé přechody odděleně. V této práci navrhujeme alternativní přímé generování spektrálních křivek na základě časové propagace náhodné vibrační vlnové funkce následované Fourierovou transformací. Nedostatek zdlouhavé a výpočetně náročné diagonalizace činí metodu vhodnou pro větší molekuly. Je zvláště vhodná pro velké Hamiltoniány, které jsou běžně získány v rámci báze harmonických oscilátorů, a algoritmus umožňuje paralelizaci. Na modelu dimeru vody jsou diskutovány základní vlastnosti konvergence. Metoda je pak použita na vibrační Ramanovy intenzity fenchonu, kde byly získány spektrální profily srovnatelné s výsledky získanými klasickými přístupy.Accurate computations of vibrational energies and vibrational spectra of molecules require an inclusion of the anharmonic forces. In standard computational protocols, a large vibrational Hamiltonian matrix is diagonalized, and spectral intensities are calculated for individual transitions separately. In this work we propose an alternate direct generation of the spectral curves based on a temporal propagation of a trial vibrational wavefunction followed by a Fourier transformation. The lack of the lengthy and computer-memory demanding diagonalization makes the method suitable for larger molecules. It is especially convenient for sparse Hamiltonians that are commonly obtained within the harmonic oscillator basis set, and the algorithm is amendable to parallelization. On a model water dimer basic convergence properties are discussed. The method is then applied to vibrational Raman intensities of the fenchone compound, were it provides spectral shapes comparable with those obtained by the classical approaches.Institute of Physics of Charles UniversityFyzikální ústav UKFaculty of Mathematics and PhysicsMatematicko-fyzikální fakult