1. The Hammerhead Ribozyme
Peter J. Mikulecky

2. What is a Ribozyme? 1) Enzyme 2) Ribonucleic Acid NOT PROTEIN
Sid Altman
Tom Cech
1989 Nobel Prize
In Chemistry

3. Structure Primary: Secondary: Tertiary:
As with proteins, we consider...
Primary:
GGCCGAACUGGUA
Secondary:
Tertiary:

4. Primary Structure
Limited to ribonucleotides (base + ribose + phosphate):
Common Bases:
A G C U
Uncommon Bases:
Pseudouridine
Inosine etc...

5. Secondary Structure Watson-Crick Base Pairing Helix Formation B-DNA
A-DNA
RNA
RNA usually assumes
A-form helices…
Small pore along helical axis
“Rungs” stack obliquely to axis

6. Secondary Structure Conserved base-pairing interactions result in...
Three “stem” regions
Uridine-containing turn
An “augmenting helix”
joining stems II and III

7. Ribozyme vs tRNAPhe
folding

8. Tertiary Structure

9. Catalytic Mechanism Intact Phosphodiester Mg2+Coordinated Transition
Cleaved
Phosphodiester

10. Catalytic Mechanism
New crystal structure shows five Mg2+ sites:

11. Ribozymes in Our Past ?

12. Ribozyme-Based Therapies
The Future of Ribozymes
In Vitro Molecular Evolution of RNA +
High Throughput Screening
Ribozyme-Based Therapies

13. In Clinical Trial... ANGIOZYME TM
Ribozyme designed to inhibit Vascular Endothelial
Growth Factor (VEGF)...
ANGIOGENESIS
HEPTAZYME TM
Ribozyme targets highly conserved sequences of the
Hepatitis C Virus...
HCV DRUG RESISTANCE

14. HIV Gene Therapy... In Clinical Trial... Bone Marrow Sample
Treat Stem Cells with Retroviral Vector
Encodes Gene for anti-HIV Ribozyme
Re-Implant Treated Cells

15. In Summary... Hammerhead Ribozyme Challenges the “Central Dogma”
Tertiary Structure Stabilized by
Conserved H-Bonding Regions
Divalent Cations Are Required for Catalysis
Ribozymes Offer Great Pharmaceutical Promise

16. References
Pley HW, Flaherty KM, and McKay DB “Three-dimensional structure of a hammerhead ribozyme” Nature 372 (1994)
Scott WG, Finch JT, and Klug A “The Crystal Structure of an All-RNA Hammerhead Ribozyme: A Proposed Mechanism for
RNA Catalytic Cleavage” Cell 81 (1995)
Scott WG, Murray JB, Arnold JRP, Stoddard BL, and Klug A “Capturing the Structure of a Catalytic RNA Intermediate: The
Hammerhead Ribozyme” Science 274 (1996)
Peracchi A, Karpeisky A, Maloney L, Beigelman L, and Herschlag D “A Core Folding Model for Catalysis by the
Hammerhead Ribozyme Accounts for Its Extraordinary Sensitivity to Abasic Mutations” Biochemistry 37 (1998)
Tuschl T, and Eckstein F “Hammerhead ribozymes: importance of stem-loop II for activity” Proceedings of the National
Academy of Sciences 90 (1993)
Heus HA, and Pardi A “Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure
Formation and magnesium ion dependence” Journal of Molecular Biology 217 (1991)
Ruffner DE, Stormo GD, Uhlenbeck OC “Sequence requirements of the hammerhead RNA self-cleavage reaction”
Biochemistry 29 (1990)
Peracchi A, Beigelman L, Scott EC, Uhlenbeck OC, Herschlag D “Involvement of a specific metal ion in the transition of the
Hammerhead ribozyme to its catalytic conformation” Journal of Biological Chemistry 272 (1997)

17. Internet References