Portland State University PDXScholar
Student Research Symposium Student Research Symposium 2013
May 8th, 9:00 AM - 10:30 AM An Alternative Ribozyme for the RNA Hypercycle
Ishak Elkhal Portland State University
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Elkhal, Ishak, "An Alternative Ribozyme for the RNA Hypercycle" (2013). Student Research Symposium. 4. https://pdxscholar.library.pdx.edu/studentsymposium/2013/Presentation/4
This Oral Presentation is brought to you for free and open access. It has been accepted for inclusion in Student Research Symposium by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. An Alternative Ribozyme for RNA Self- Assembly
Ishak Elkhal May 8, 2013 Portland State University, Department of Chemistry – Lehman Lab Formation of the Earth → Life
Joyce (2002) Nature 418, 214-221 Formation of the Earth → Life
Joyce (2002) Nature 418, 214-221 Formation of the Earth → Life
Joyce (2002) Nature 418, 214-221 Development and Diversification of life The RNA world
Joyce (2002) Nature 418, 214-221 Lehman Lab Approach: Recombination
Symmetric Lehman Lab Approach: Recombination
Symmetric
Asymmetric Lehman Lab Approach: Recombination
Inactive
Adapted from Hayden & Lehman (2006) Chem. Biol. 13, 909-918. A novel Ribozyme for Self-Assembly: CX Organismal source: Coxiella burnetii Group I intron (Self-Splicing RNA) in 23S rRNA
Raghaven et al. (2007) J. Bacteriol 189, 6572-6579. A novel Ribozyme for Self-Assembly: CX Organismal source: Coxiella burnetii Group I intron (Self-Splicing RNA) in 23S rRNA
CX Azoarcus 266-nt 198-nt
Raghaven et al. (2007) J. Bacteriol 189, 6572-6579. A novel Ribozyme for Self-Assembly: CX Organismal source: Coxiella burnetii Group I intron (Self-Splicing RNA) in 23S rRNA
CX Azoarcus 266-nt 198-nt ~38% GC ~70% GC
Raghaven et al. (2007) J. Bacteriol 189, 6572-6579. A novel Ribozyme for Self-Assembly: CX Organismal source: Coxiella burnetii Group I intron (Self-Splicing RNA) in 23S rRNA
CX Azoarcus 266-nt 198-nt ~38% GC ~70% GC 4-nt IGS 3-nt IGS
Raghaven et al. (2007) J. Bacteriol 189, 6572-6579. Recombination
Adapted from Riley & Lehman Hayden & Lehman (2006) “Drawing Board” (2013). (2003) Chem. Biol. 10, 1233-1243. Chem. Biol. 13, 909-918. U A A A C U A G
IGS “Internal Guide Sequence” Internal Guide Sequences (IGS) seeks out the “Tag” 5’ G U A G 3’ | | |
3’ U A U C 5’
C U U A
U C A U U A A A C U A G
IGS “Internal Guide Sequence” Internal Guide Sequences (IGS) seeks out the “Tag” 5’ G U A G 3’ | | |
3’ U A U C 5’
C U U A
U C A U Steps towards getting a self- assembling CX ribozyme 1. Check functionality post-modification
2. Optimize reaction conditions
3. Kinetic characterization
4. Cleavage and self-assembly
Functionality Expected: S + E → E*S
← CX*S ← CX
← Substrate Functionality Expected: S + E → E*S
← CX*S ← CX
It works!
← Substrate Optimization
Assay for [MgCl2] Negative control Assay for cation identity ↓
← Increasing [MgCl2]
Optimal Conditions: MgCl2 at 40 mM Optimization
Assay for Temperature and Time Assay for pH Negative control ↓
Increasing pH →
o Optimal Conditions: 40 mM MgCl2, pH = 7.5, 37 C, 2 hr I83 4
3.5
3 Kinetics 2.5 2 I87-1 1.5 1 4 PercentReacted
0.5 3 0 0 50 100 150 200 250 300 2 Time (min) 1
PercentReacted I87-2 0 0 500 1000 1500 2000 3.5 Time (min) 3 2.5 I91-1 2 1.5 6
1
5 PercentReacted 0.5 4 0 3 0 500 1000 1500 2000 2 Time (min)
1 PercentReacted 0 0 500 1000 1500 2000 0.668 uM Time (min) 7 6 5 4 3 2 1 0 0 500 1000 1500 2000 Self-assembly
Partial PCR of CxY and CxX fragments
Full length product is usually here → (266-nt)
164-nt, Y 142-nt, X Conclusions and Future Work
• The modified CX intron functions as a ribozyme
• The optimal reaction conditions are: o • 40 mM MgCl2, Tris-HCl (pH = 7.5), 37 C for 2 hours
• The ribozyme efficiently undergoes a partial PCR and is ready to be subjected to the self-assembly
• Speculative: The ribozyme is ≤ ½ as fast as the Azoarcus system Acknowledgments
• Lehman Lab • Dr. Niles Lehman • Jessica Yeates • David Gofreed
• Friends and Family • Thank you for your support!
• NASA