Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA

1. Optimization of non-natural nucleotides for selective incorporation oppositedamaged DNA Diana Vineyard,Xuemei Zhang,Alison Donnelly,Irene Leeand Anthony J. Berdis Org. Biomol. Chem., 2007, 5, 3623–3630

2. Nucleic acid bases :Purines and Pyrimidines Purines Adenine Guanine Pyrimidines Uracil (RNA) Cytocine Thymine (DNA)

3. Structure of DNA : Bases linked to Sugar-Phosphate backbone Phosphodiester linkage

4. Base Pairs: H-bonding between Purines and Pyrimidines

5. Structure of DNA : Double Helix Phosphates and Sugars: Hydrophilic Bases: Hydrophobic H – Bonding between Base pairs

6. Base Stacking Interactions • Hydrophobic stacking interactions in which two of more bases are positioned with the planes of their rings parallel • Also involve combination of van der Waals an dipole-dipole interactions between bases • Help to minimize contact with water and are very important in stabilizing 3D structure of DNA

7. DNA damages Oxidative damage UV induced damage: Thymine dimers Hogg, M.; Wallace, S. S.; Doublie, S. Current Opinion in Structural Biology 2005, 15, 86–93

8. DNA damages: Can be mutagenic if bypassed by DNA polymerase Normal C-G pair 8-oxoG –dC (dexoycytosine 3’-monophosphate) Mutagenic ! 8-oxoG –dA (dexoyadenosine 3’-monophosphate) Hogg, M.; Wallace, S. S.; Doublie, S. Current Opinion in Structural Biology, 2005, 15, 86–93

9. Abasic Sites • Most freaquent DNA lesion encountered by cells (~10,000 per human cell per day) • Produced by hydroxyl radical attack on the sugar residue releasing the free base or spontaneous depurination Hogg, M.; Wallace, S. S.; Doublie, S. Current Opinion in Structural Biology, 2005, 15, 86–93

10. DNA Polymerases • Belong to different families • Polymerase domain is composed of three sub domains: fingers, palm and thumb • The exonuclease domain increases proof- reading activity by10-100 fold

11. RB69 DNA Polymerase gp43:a close relative of T4 DNA polymerase gp43

12. DNA Replication • Substrate : deoxynucleoside 5’-triphosphates or dNTP (dATP, dGTP,dTTP,dCTP) dATP

13. Chemistry of DNA replication Pyrophosphate ion

14. Can abasic sites be bypassed by DNAPolymerase? Primer extended by DNA polymerase Primer not extended RB69 gp43 exo- is able to extend a primer by incorporating dAMP, and to a lesser extent dGMP, opposite an abasic site. Hogg, M.; Wallace, S. S.; Doublie, S. The EMBO Journal, 2004, 23, 1483–1493

15. Synthetic base analogs

16. Goal: translesion DNA synthesis Translesion DNA synthesis: The ability of a DNA polymerase to incorporate opposite a DNA lesion DNA substrate (primer): X= abasic site - part I or A, C, G, T (templating bases) – part II DNA polymerase: T4 DNA polymerase (gp43 exo-)

17. Surface-ionization potentials for each nucleobase Red: electronegative Green: neutral Blue: electropostive

18. dNTP analogs • 5-cyanoindolyl-2’-deoxyriboside 5’-triphosphate (5-CyITP) • 5-ethylindolyl-2’-deoxyriboside 5’-triphosphate (5-EtITP) • 5-ethyleneindolyl-2’-deoxyriboside 5’-triphosphate (5-EyITP) • 5-methylindolyl-2’-deoxyriboside 5’-triphosphate (5-MeITP)

19. Parameters Kd:The first kinetic step represents binding of dXTP to the polymerase–DNA complex Kpol: conformational change Kchem: The final stage of the catalytic cycle is the phosphoryl transfer step that is required for elongation of the primer strand Kobs: observed product formation

20. What physical properties dictate the incorporation of unnatural nucleotides opposite abasic sites?

21. Part I:Enzymatic incorporation opposite an abasic site Rate constants (Kobs) for the incorporation of 5-CyITP opposite an abasic site on as a function of nucleotide concentration Rate constants (Kobs) vs. [5-CyITP] Hyperbolic; Fitted to Mechaelis-Menten kinetics

22. Part I: Comparison of 5-CyITP with 5-NITP: oberservations • 5-NITP: 5-nitroindolyl-2’-deoxyriboside 5’-triphosphate • Note: two analogs are similar in size, solvation energies, dipole moments, and presence of π-electron density • kpol of 29 s−1 for 5-CyITP is ∼4-fold slower than the kpol value of 126 s−1 for • Kd for 5-CyITP (58μM) is 3-fold higher than 18 μM for 5-NITP • 5-NITP more efficiently incorporated?

23. Comparison of 5-CyITP with 5-NITP: explanations for Kpol • The nitrogen atom of the nitro substituent group of 5-NITP possesses a partial positive charge and therefore can interact with O4 of Thymine adjacent to abasic site(dipole-dipole interactions favor base stacking) * *

24. Comparison of 5-EyITP with 5-CEITP: observations • 5-CEITP: 5-cyclohexylindolyl-2’-deoxyriboside 5’-triphosphate • 5-EyITP binds10-fold more weakly than 5-CEITP • The faster kpol value of 94 s−1 for 5-EyITP compared to 25 s−1 for 5-CEITP • 5-CEITP more efficiently incorporated?

25. Comparison of 5-EyITP with 5-CEITP: explanations • Reduced size and hydrophobicity of 5-EyITP limits its ability to adequately displace any water molecules (faster Kd) that may occupy the void of the abasic site as effectively as the larger 5-CEITP • The faster kpol value of 94 s−1 for 5-EyITP compared to 25 s−1 for 5-CEITPlikely reflects the ability of the smaller analog to facilitate the conformational change preceding phosphoryl transfer more effectively than the larger 5-CEITP analog

26. Part I: Conclusion Nucleotide incorporation opposite a non-templating DNA lesion can occur via enhanced base-stacking interactions caused by reductions in solvation energies and proper steric arrangements rather than through overall increases in πelectron surface area

27. Part I: Conclusion How about π-electron surface area?

28. Part I: Conclusion – how to design selectivity of incorporation Selectivity for incorporation opposite an abasic site can be modulated through simple alterations to the π-electron surface area of a non-natural nucleotide Data fit parabolic function well

29. Part II:Enzymatic insertion opposite templating nucleobases • All small non-natural nucleotides synthesized in this study are poorly incorporated opposite any templating nucleobase

30. Selectivity of incorporation with respect to of a non-natural nucleotideopposite a templating base Catalytic efficiency vs. π-electron surface area: Parabolic curve is non-existent

31. Part II : Conclusions • Challenging to rationally design a nucleotide for selective incorporation opposite a templating nucleobase • Difficult to find a model explaining kinetic behavior of these non-natural nucleotides incorporated opposite templating nucleobases • Nucleotide incorporation opposite templating DNA is influenced by these interrelated biophysical features • Abasic site appears to reduce the overall complexity of these interactions such that the catalytic efficiency for incorporation is primarily influenced by only the size and π-electron density of the incoming nucleotide

32. Significance and future work • Designing new base analogs to control DNA polymerase activity – applying synthetic organic chemistry • More accurately define factors that control incorporation opposite a templating bases – physical chemistry • Better understand mechanism of DNA repair – DNA lesions play key role in cancer development

33. Acknowlegement • Dr. Martin Case • All the Chem 258 students