1. AMBER Parameters for Pseudouridine Delon Wilson Advisor: J. SantaLucia

2. Outline  Introduction and Motivation  Pseudouridine & Modified Nucleic Acids  Atomic charge in MM  RESP/ Method  The MEP  Charge Fitting with Restraints  Results  Conclusion

3. Nucleic Acid Structure Base Sugar Phosphate G,C,A,T (DNA) G,C,A,U (RNA) Deoxyribose (DNA) Ribose (RNA)

4. PSU and Modified NA’s  Found as natural substances or obtained synthetically  Pseudouridine – 1% in rRNA, tRNA  produced by chemically modifiying one of the four bases (G, C, A, and T in DNA or U in RNA)  Important in biochemical regulation  Used extensively in chemistry, biochemistry, and pharmacology as probes to study biological mechanisms

5. PSU and Modified NA’s  Major cause of spontaneous mutation in E. coli results from the presence of an unusual base in the DNA. e.g. 5-MethylCytosine  Successful applications as antibiotics or chemotherapeutic agents  e.g. AZT interferes with the replication of HIV (Human Immunodeficiency Virus)

6. Molecular Mechanics  Modeling of biological systems  Accurate representation of electrostatic interactions crucial for force field application  Suitable force field parameters required for molecular mechanics and dynamics  force constants, atom types, bond distances, atomic charges

7. Molecular Mechanics  Amber: A suited of programs developed by Peter Kollman & Coworkers at UCSF  Force field referred to by same name  Parameters for regular NA’s (A,C,T,G,U) developed  Suitable parameters for modified NA’s not available  An albatross to computations for systems involving substantial amounts of mod. NA’s

8. Potential Energy Model The force field energy where

9. Pseudouridine Base joined to ribose via C-C, Vs. C-N in regular NA’s Uridine Psu

10. Starting structure from PDB Perform Geometry Optimization (HF-631G*) Compute Electrostatic Potential Charges (pop=mk) Fit ESP charges (RESP) MD simulation (AMBER)

11. Initial Structure

13. Gaussian Keywords  #p hf 6/31-g(d) opt pop=mk geom=connectivity test iop(6/33=2)  Duration: around 5hrs

14. ESP: Some Common Methods  Mulliken Population Analysis-does not reproduce ESP closely enough  Natural Population Analysis`  ESP derived using CHelpG scheme  ESP derived using MKS scheme

15. Calculation of ESP/MEP  Calculate approximate Ψ from eq geom  Calculate e density from psi  The ESP at point 1 is:

16. RESP  Least squares algorithm derives atom centered charges that best reproduces MEP  Potential calculated on large number of points on 4 shells of surfaces defined by {1.4, 1.6, 1.8, 2.0} x VDW radii  ESP at each point derived from QM 

17. Results.4312 (.4295) -.6135 (-.6223).0748 (.0679).0748 (.0679).0264 (.0558).0788 (.1065).1171 (.1174) -.3604 (-.3548).0461 (.0615).2808 (.2022).0736 (.0670).0974 (.0972) -.5962 (-.6139).4098 (.4186).3885 (.4376) -.6271 (-.6541).0303 (-.1081).1714 (.2405) -.5332 (-.6230).3842 (.8922).0069 (-.5317) -.0759 (.3896).2104 (.1226).5173 (.9290) -.5841 (-.6096) -.3407 (-.6770).3418 (.4311) -.2671 (-.7255).3402 (.3859) Partial charges on pseudouridine: values in parenthesis from Amber website, std=0.23

18. Results  Calculations performed on a single Pentium IV processor.  Average CPU time to perform the geometry optimization of each nucleoside in the order of several hours (~5)  Charges for each ribonucleotide are in a good agreement with AMBER standard reference file (all_nuc94.in).  Calculations on pseudouridine deviate more than expected from the contributed values provided at the AMBER website.

19. …To do  Investigate the dependence of the charges on conformation  Determine force field parameters for all (~103) of the naturally occurring modified nucleotides that occur in RNA and DNA.  Extend the AMBER force field so that nuclei acids with modifications may be routinely modeled.  Develop a novel force field specifically tailored to nucleic acid applications (NA_FF).

20. References/Acknowlegment  U. C. Singh and P. A. Kollman J. Comp. Chem. vol.5, no.2, 129-145 (1984)  B.H. Besler, K.M. Merz Jr., and P.A. Kollman J. Comp. Chem. vol.11, no.4, 431-439(1990)  JSL Lab  Schlegel Lab

21. Questions etc … Thank you