The Schlick Group Retreat
February 2008
Two Approaches of RNA Design:
Fluorescent Riboswitch & Ligase Selection
Namhee Kim and Giulio Quarta
Laboratory of Prof. Tamar Schlick
1. Design and Applications of Novel RNAs
2. Novel Candidates of Fluorescent Riboswitch
3. Pool Design & Selection of Ligase Ribozymes
4. Future Directions
1. RNA Design
 Certain RNA aptamers have been found to bind and function
with specific compounds and molecules
 Examples:
– In vitro: Malachite Green Aptamer (MGA), Hammerhead Ribozyme
– In vivo: Thiamine Pyrophosphate Binding Riboswitches (TPP-BR)
 RNA Design: Allosteric shifts can prevent/stabilize
secondary/tertiary structures upon ligand binding
Baugh et al., J. Mol. Biol. 301:117 -128 (2000)
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Serganov et al., Nature 441:1167 -1171 (2006)
NYU/BIOMATH
Gene Regulation by TPP Riboswitch
Translation initiation
(thiM genes), with the
dissociation of TPP
Transcription termination
regulation (thiC genes),
with dissociation of TPP
Serganov et al., Structural basis for gene regulation by a thiamine pyrophsphatesensing riboswitch Nature 441:1167 -1171 (2006)
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2.1. Modular Design
Trans.
OFF
NO
FLUR.
+
And
T2
T1
FLUR.
Trans.
ON
+
M1
T3
4
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2.2. Co-transcriptional Folding
of TPP Riboswitch
125nt: Thi-box formed
150nt: a helix formed
175nt: TPP domain is disappeared
and anti-terminator formed
185nt: TPP domain is reformed and
terminator formed
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Thermodynamic Analysis
C 2-4 (a) S1
C 2-4 (b) S1
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Experimental Results
Form 2
State 2
DNA template
TTP
Malachite Green
2-1
-
- + +
+ - +
2-2
- - +
- + -
2-3
+ + -
- + +
+ - +
-
2-4
A1-TPP fusion
- + +
+ - +
-
- +
+ -
+
+
6% gel, chase by 100mkM NTPs
No Structural Change Upon TPP (2-1, 2-2, 2-3, 2-4)
Structural Change Upon TPP (Wild Type: A1-TPP fusion)
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Provided by the Nudler lab
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Wild-Type Riboswitch Landscape
TPP
State1:
Antiterminator
No TPP
binding
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State2:
Terminator
Yes TPP
binding
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New Candidate Sequence (Terminator added)
TPP
C 2-4
State1:
Anti-terminator
No TPP binding
10 Yes MG binding
State2:
Terminator
Yes TPP binding
No MG binding
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3.1. Pool Design for RNA Ligases
Requirement: Sequence length ≤ 60 nt
Pool 1: 4_2 + 5_2
Motif ID: 31
66% of Seq 8, Matrix 13
AGCGCCGUGGCAGGGCUCAUAACCCUGAUGUCC
UCGGAUCGAAACCGAGCGGCGCUACCA
34% of Seq 12, Matrix 8
GGCAGUACCAAGUCGCGAAAGCGAUGAUGGUAA
CCUUGCAAAGGGUUAAGCUGCC
Pool 2: 4_1 + 5_1
6% of Seq 5, Matrix 4
GGCAGUACCAAGUCGCGAAAGCGAUGGCCUUGCA
AAGGGUAUGGUGCUGCC
94% of Seq 12, Matrix 11
GGCAGUACCAAGUCGCGAAAGCGAUGAUGGUAAG
CCUUGCAAAGGGUUAAGCUGCC
Pool 3: 4_1 + 4_2
Motif ID: 42
77% of Seq 6, Matrix 13
GGGACGAGCACGUGAAUCGUCUCGACGUGUGUA
GGGGAAAGUAUCCCCCGUCCC
23% of Seq 25, Matrix 12
GCCCCGCUGAUGAGGUCAGGGAAGACCGAAAGU
GUCGACUCUACGGGGC
Pool 4: Random pools with 60nt
Emergence of a fast-reacting ribozyme that is capable of
undergoing continuous evolution, Sarah B. Voytek and Gerald
F. Joyce, PNAS, (2007)
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100% of Seq 8, Matrix 4
AGCGCCGUGGCAGGGCUCAUAACCCUGAUGUCC
UCGGAUCGAAACCGAGCGGCGCUACCA
Designed by RAGPOOLS
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3.2. Selection of RNA Ligases
 We are scanning 109 sequences in designed pools 1, 2,
3, and random pool using RNAMotif with consensus of
DSL ligase and T80 secondary motif without sequence
restriction
 But we did not get positive results yet in designed pools
 We try to start with known sequences (DSL and T80)
and use 22 mixing matrices
 Preliminary Scanning Results …
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4. Future Directions
 Fluorescent riboswitch:
Optimize the sequence to have the ideal structural
changes and two clear clusters in the energy
landscape, in vitro and in vivo experimental
verification
 Ligase selection:
Design pools starting with known or other sequences
and provide a proof that designed pools are efficient,
expanding in silico pool size into 1015
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