Lukasz Bielecki, Ph. D.
lucas.bielecki@gmail.com

Conformational Analysis of Modified DNA and RNA by Methods of Molecular Dynamics Simulation

Summary of PhD thesis written in October 1999 under supervision of Prof. Dr. Ryszard W. Adamiak

Laboratory of Structural Chemistry of Nucleic Acids
Institute of Bioorganic Chemistry of the Polish Academy of Sciences
Noskowskiego 12/14, PL-61-704 Poznan

INTRODUCTION
Over the last decade, molecular modelling has become a method widely accepted in structural chemistry and structural molecular biology. Various simulation techniques exist for objects of a wide range of molecule size. Among those, molecular dynamics (MD) simulation methods have proved their applicability for elucidating the structure and dynamics of biopolymers, both when used in the final stages of studies accomplished by NMR or X-Ray methods, and as an independent strategy of conformational analysis directed for objects not yet synthesised. For this latter approach, the problem of the reliability of simulation protocols and representation of the molecular system is crucial.
The main aim of my PhD thesis was conformational analysis of short RNA and DNA fragments containing non-standard structural elements in order to evaluate their influence on the local and global duplex conformation using various strategies of molecular dynamics simulations as well as a variety of procedures for structural analysis and visualization of the results.

METHODS
All simulations were performed using the Amber forcefield implemented into the Discover (MSI) and Sander (UCSF) programs. Model building procedures available in the InsightII (MSI) and AMBER 4.0 (UCSF) suites of programs were used. For parametrisation of the modified nucleosides, the Gaussian94 and Resp (UCSF) programs were applied. The experiments were performed using the advanced strategies of in aqua simulations: both the nonbonded cutoff approach and the one including the PME method for treatment of nonbonded interactions. The simulation time varied from 200 to 1000 ps, dependent on the complexity of the system. The simulations were carried out on two CRAY machines (YMP-EL and J-916) working in the Poznan Supercomputing and Networking Center affiliated to our Institute. Subsequent conformational analysis of the MD trajectories has been performed on the CRAY and SGI machines using several programs, e.g. CURVES 5.1 (Lavery & Sklenar), SAHN (program for cluster analysis applied for evaluating the internal conformational similarity within the trajectories) as well as our own procedures, mostly designed for the InsightII environment, e.g. to monitor the stacking interactions within the nucleic strands or to evaluate the nucleic acid-solvent interactions. Other procedures were created for visualisation of the resultant structures as well as for presentation of structural parameters.

DNA DUPLEXES CONTAINING 1,N(6)-ETHENOADENINE
A series of 11 bp DNA duplexes containing a single 1,N(6)-ethenodeoxyadenosine (edA) residue in the middle position of the molecule was examined. Two cases were selected where the modified nucleo-base was involved in stable hydrogen bond formation with the opposite base. In the syn-edA - anti-dC case, one hydrogen bond was identified over the considerable part of the trajectory. In the other case, two stable hydrogen bonds were found between syn-edA and anti-dG, which conformed previous NMR and X-Ray data. For this model and for the analoguous unmodified duplex with the dC-dG pair, detailed in aqua MD simulations were performed. The stability of the trajectories obtained clearly confirmed the better reliability of the PME method over the nonbonded cutoff approach. The resultant structures showed the ability of the modified residue to adapt to the B-DNA duplex conformation by forming a base pair with the opposite guanosine, which mimics a standard Watson-Crick pair. The approach based on the cluster analysis method applied to the trajectory conformations made it possible to identify typical conformations which seem to predominate during the simulation.

a-ANOMERIC NUCLEOSIDE IN A DNA DUPLEX
Little is known about the influence of a single a-nucleoside residue on the overall conformation of a DNA duplex. Our special interest was drawn by the question why nature had “chosen” only b-nucleosides as the structural elements of all known nucleic acids. I performed a series of in vacuo and in aqua molecular dynamics simulations in order to test the hypothesis of a duplex axis bending caused by such a modification, which was suggested by certain physical analyses of molecules of this kind. Paralelly, these experiments helped me to optimise the simulation protocols used in my research. Preliminary simulations (especially in vacuo and those including the nonbonded cutoff approach) caused various kinds of massive distortions within the objects tested (both unmodified and a-anomer-containing DNA). In most cases, however, the duplexes modified with a single a-anomeric nucleoside (a-adenosine or a-1,N(6)-ethenodeoxyadenosine) showed
a considerable degree of main axis bending when compared to the reference (unmodified) molecules. Still, the most advanced experiments including the PME method have not provided a doubtless answer as to the existence of such an effect. On the other hand, I have found a clear ability of the a-anomeric residue to form base pairs with the opposite
b-nucleosides, which mimic the respective beta-beta pairs, both in the molecules containing dA-dT and edA-dG pairs.

DNA DUPLEXES CONTAINING THE LUMINARINE FLUOROPHORE
An experiment was performed to describe the conformational dynamics of a DNA duplex containing a single deoxyluminarosine residue (the strongest fluorophore applied in our group as a conformational probe for structural research). It suggested that this three-ring system easily adapts itself to the stacking interactions within the DNA strand and, to some extent, it is also capable of forming hydrogen bonds with opposite guanosine and thymidine.

BULGE RNA DUPLEXES MODIFIED WITH 2-AMINOPURINE
As part of a larger project aimed at elucidating the structure and dynamics of short RNA duplexes containing purine bulges, I performed molecular dynamics experiments in order to simulate the conformation of such molecules containing adenosine or 2-aminopurine riboside (its fluorescent isomer) residues in the bulge region. The in aqua experiments, both using the cutoff and PME protocol, resulted in trajectories which suggested a considerable bending of the bulged duplex, whereas the bulge residues tend to adopt an orientation towards the solvent. No difference between the behaviour of the
2-aminopurine system compared with the native adenosine containing bulge was observed. This confirms earlier thermodynamical results from our group concerning the behaviour of 2-aminopurine substituted for adenine.

HIV-1 TAR RNA AND ITS MODIFICATIONS
Experiments focused on the HIV-1 TAR RNA molecules were aimed at identifying the conformational dynamics within the unpaired region of this molecule. I performed three PME in aqua MD simulations of molecules differing with the sequence of the apical loop, which started from the same initial conformation acquired from PDB (conformation No. 17 selected from the 1ANR set provided by the Varani group). One molecule was consistent with the native TAR RNA sequence and two others were modified with 2-aminopurine in the G34 and A35 positions, respectively. The resultant trajectories helped me to describe the dynamics of the residues within the loop and suggested that 2-aminopurine can replace adenine, but not guanine, with little effect on the local structure. In contrast to the NMR data, the molecular dynamics experiment shows a strucuralization of the apical loop. The final conformation of the unmodified molecule was also used for an additional molecular mechanics analysis of the probability of magnesium cation binding near the trinucleotide -UCU- loop. This experiment showed two sites of possible binding of Mg2+.

RNA HYDRATION
The trajectories of the experiments described above which concerned RNA molecules were also analysed in order to identify the specific hydration pattern, especially for the unpaired regions of RNA. I calculated the relative hydration of particular atoms, including hydrogens from C-H groups. This analysis was correlated with the conformation of unpaired regions of the molecules, especially in regard to the tendency of nucleosides to adopt orientation towards the inside or outside of the molecule. I have also identified several water bridges linking highly hydrated atoms on the surface of the bulged regions of the RNA molecules.

CONCLUSIONS
The simulations presented in my PhD covered a wide variety of problems which may be effectively researched using the molecular dynamics simulation methods. The detailed results of the experiments performed have so far been published in the following papers:

1. T. Kulinski, M. Olejniczak, H. Huthoff, L. Bielecki, K. Pachulska-Wieczorek, A. T. Das, B. Berkhout & R. W. Adamiak: The Apical Loop of the HIV-1 TAR RNA Hairpin Is Stabilized by a Cross-loop Base Pair. J. Biol. Chem. (Vol. 278 (40)) 2003, pp. 38892-38901.

2. M. Olejniczak, Z. Gdaniec, A. Fischer, T. Grabarkiewicz, L. Bielecki, R. W. Adamiak: The bulge region of HIV-1 TAR RNA binds metal ions in solution. Nucleic Acids Res. (Vol. 30) 2002, pp. 4241-4249.

3. T. Kulinski, L. Bielecki, R. W. Adamiak: Structure and dynamics of adenosine loops in RNA bulge duplexes as revealed by linked application of thermodynamics, spectrofluorimetry and simulation of molecular dynamics. Nucleic Acids Res. Supplement (No. 1) 2001, pp. 139-140.

4. L. Bielecki, R. W. Adamiak: Structure and dynamics of DNA duplexes containing single a-adenosine residues. Acta Biochimica Polonica (Vol. 48) 2001, pp 103-111.

5. L. Bielecki, B. Skalski, I. Zagorowska, R. E. Verrall & R. W. Adamiak: Fluorescent a-anomeric 1,N(6)-ethenodeoxyadenosine in DNA duplexes. The a-edA / dG pair. Nucleosides, Nucleotides & Nucleic Acids (Vol. 19) 2000, pp. 1735-1750.

6. L. Bielecki, T. Kulinski & R. W. Adamiak: Structure and dynamics of adenosine loops in RNA bulge duplexes. RNA hydration at the bulge site. In: RNA Biochemistry and Biotechnology. J. Barciszewski and B. F. C. Clark (eds.), NATO ASI Series, Kluwer Academic Publishers 1999, pp. 73-87.

7. T. Kulinski, L. Bielecki, M. Olejniczak, I. Zagorowska & R. W. Adamiak: Structure and dynamics of the apical loop region of
29-mer hairpin of the TAR RNA HIV-1 sequence. Collection Symposium Series (Vol. 2) 1999, pp 191-196.

8. A. Fischer, Z. Gdaniec., M. Olejniczak, L. Bielecki, R. W. Adamiak: Does 29-mer RNA hairpin of the HIV-1 TAR RNA sequence bind magnesium? Nucleic Acids Symposium Series  (Vol. 42) 1999, pp. 117-118.

9. T. Kulinski, L. Bielecki, I. Zagorowska & R. W. Adamiak, R. Rigler: Dynamics of RNA bulge duplexes. 2-Aminopurine labelled adenosine loops. Spectroscopy of Biological Molecules: Modern Trends. Annex, 1997 UNED Madrid, Spain, pp. 39-40.

10. T. Kulinski, L. Bielecki, I. Zagorowska & R. W. Adamiak: Introductory data on dynamics of RNA bulge duplexes. 2-Aminopurine labelled adenosine loops. Collect. Czech Chem. Commun. (Vol 61) Special Issue 1996, pp. 265-267.

11. R. W. Adamiak & L. Bielecki: An optimized protocol for in vacuo molecular dynamics simulation and trajectory analysis of modified DNA duplexes. Computational Meth. Sci. Tech. (Vol. 2) 1996, pp. 7-16.


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