SUPPORTING INFORMATION Tuning RNA folding and function through rational design of junction topology May Daher1,†, Anthony M. Mustoe2,‡,†, Alex Morriss-Andrews2,3,†, Charles L. Brooks III2,3*, and Nils G. Walter1* 1
Single Molecule Analysis Group and Center for RNA Biomedicine, Department of
Chemistry, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 481091055, USA 2Biophysics, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109-1055, USA 3
Department of Chemistry, University of Michigan, 930 N. University Avenue, Ann Arbor,
MI 48109-1055, USA †
These authors contributed equally
*To whom correspondence should be addressed. Email:
[email protected] or
[email protected] ‡
Present address: Department of Chemistry, University of North Carolina Chapel Hill,
NC 27599-3290, USA
Figure S1. Docking probabilities of HHHH, HS7H, HCH, and HHH junctions possessing shortened or lengthened A and/or B helices. Probabilities are shown relative to the HHHH junction. The probabilities for the HHHH, HS7H, and HHH junctions are repeated from Figure 1 and are included for reference (and highlighted via coloring).
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Table S1: Sequences used in TOPRNA simulations. Models were built as a single strand using the toprnaCreate.pl Perl utility. Note that TOPRNA has minimal dependence on sequence, and hence sequences of inserted elements (e.g. loops, helices, etc.) are arbitrary.
Construct Sequence HHHH H-1HHH HHHH-1 H-1HHH-1 HHCHH HHHHS2 HS2HHH HS4HHH HHHS2H HHHS4H H+1S7H HS7H+1 H+1S7H+1 H-1S4HS1 HS4H-1S1 HS3H HS6H HS7H HS8H HS9H HS10H H-1S8HS1 HS8H-1S1 HCH
GGAGAGAGAAGUCAACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUAC GGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUGACAGUCCUCUCC GGAGAGAGAAGUCACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUACG GGGGAAACUUCGUGGUGGCCGAAAGGCCACCGACAGUCCUCUCC GGAGAGAGAAGUCACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGACGG GGGAAACUUCGUGGUGGCCGAAAGGCCACCUGACAGUCCUCUCC GGAGAGAGAAGUCCCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGACGGG GGAAACUUCGUGGUGGCCGAAAGGCCACCGACAGUCCUCUCC GGUGGCCGAAAGGCCACCUGACAGUCCUCUCCUUUUGGAGAGAGAAGUCAACC AGAGAAACACACGGAAACGUGGUAUAUUACCUGGUACGGGGGAAACUUCGU GGAGAGAGAAGUCAUUACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGU ACGGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUGACAGUCCUCUCC GGAGAGAGAAGUCAACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUAC GGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUUUGACAGUCCUCUCC GGAGAGAGAAGUCAACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUAC GGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUUUUUGACAGUCCUCUCC GGAGAGAGAAGUCAACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUUU ACGGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUGACAGUCCUCUCC GGAGAGAGAAGUCAACCAGAGAAACACACGGAAACGUGGUAUAUUACCUGGUUU UUACGGGGGAAACUUCGUGGUGGCCGAAAGGCCACCUGACAGUCCUCUCC AAAUAGAGAAGCGCAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACA UUACCUGGUACCCCCUGCGCAGUCCUAUUU AAAUAGAGAAGCGAACCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACA UUACCUGGGUACCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGCAACCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUAC AUUACCUGGGUACCCCCUGCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCUCGCAGUCCUAUUU AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACCCCCUCGCAGUCCUAUUU UCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAU UUUUUUGGCGUGGUACAUUACCUGGU
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H-1SCHS1 UCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAU UUUUUUGGCGUGGUACAUUACCUGGU HSCH-1S1 UCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAU UUUUUUGGCGUGGUACAUUACCUGGU H+1CH UGCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGCAACCAGAGAAACACACGCCAA AUUUUUUUGGCGUGGUACAUUACCUGGU HCH+1 UCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGAACCCAGAGAAACACACGCCAAA UUUUUUUGGCGUGGUACAUUACCUGGGU H+1CH+1 UGCGCAGUCCUCUCCUUUUAAAUAGAGAAGCGCAACCCAGAGAAACACACGCCA AAUUUUUUUGGCGUGGUACAUUACCUGGGU H-1S7H AAAUAGAGAAGCGACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUU ACCUGGUACCCCCCGCAGUCCUAUUU HS7H-1 AAAUAGAGAAGCGACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUU ACCUGGACCCCCUCGCAGUCCUAUUU H-1S7H-1 AAAUAGAGAAGCGCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUUA CCUGGACCCCCCGCAGUCCUAUUU HS7HS2 AAAUAGAGAAGCGAUUACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUAC AUUACCUGGUACCCCCUCGCAGUCCUAUUU HHH AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUU H+1HH AAAUAGAGAAGCGCAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACA UUACCUGGUACGCGCGCGUUUUCGCGCGCGUUGCGCAGUCCUAUUU HHH+1 AAAUAGAGAAGCGAACCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACA UUACCUGGGUACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUU H+1HH+1 AAAUAGAGAAGCGCAACCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUAC AUUACCUGGGUACGCGCGCGUUUUCGCGCGCGUUGCGCAGUCCUAUUU HS2HH AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACGCGCGCGUUUUCGCGCGCGUUUUCGCAGUCCUAUUU HS4HH AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACGCGCGCGUUUUCGCGCGCGUUUUUUCGCAGUCCUAUUU HHS2H AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUUUACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUU HHS4H AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUUUUUACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUU HCHH UCGCAGUCCUAUUUUUUUAAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAU UUUUUUGGCGUGGUACAUUACCUGGUACGCGCGCGUUUUCGCGCGCGU HHCH ACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUUUUUUAAAUAGAGAAGCG AACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUUACCUGGU H-1HH AAAUAGAGAAGCGACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUU ACCUGGUACGCGCGCGUUUUCGCGCGCGUCGCAGUCCUAUUU HHH-1 AAAUAGAGAAGCGACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUU ACCUGGACGCGCGCGUUUUCGCGCGCGUUCGCAGUCCUAUUU H-1HH-1 AAAUAGAGAAGCGCCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAUUA CCUGGACGCGCGCGUUUUCGCGCGCGUCGCAGUCCUAUUU HHHHH AAAUAGAGAAGCGAACCAGAGAAACACACGCCAAAUUUUUUUGGCGUGGUACAU UACCUGGUACGGCGUUUUCGCCGUUUCGCGCUUUUGCGCGUUUCGGCGUUUUC GCCGAUCGCAGUCCUAUUU
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Table S2: Compilation of prior experimental studies of the hairpin ribozyme junction and comparison to TOPRNA results. TOPRNA and experimental ∆∆Gdock values are computed relative to the HHHH topology, as described in the main text. TOPRNA Pdock values are are normalized to the HHHH topology. Experimental krel values are catalytic rate constants normalized to the junction indicated in parentheses. [Mg2+]1/2 is the magnesium concentration of half-maximal docking. trFRET: time resolved FRET. Junction
TOPRNA
Experimental
Reference key
HHHH
Pdock = 1.0 (∆∆Gdock = 0.0 kJ/mol)
∆∆Gdock = 0.0 kJ/mol (6) ∆∆Gdock = 0.0 kJ/mol (19) [Mg2+]1/2 = 30 M (16)
(6) Equilibrium trFRET (19) Equilibrium trFRET (16) Equilibrium bulk FRET
HCH
Pdock = 0.19 (∆∆Gdock = 4.1 kJ/mol)
∆∆Gdock = 5.7 kJ/mol (6) [Mg2+]1/2 = 25 mM (16) krel(HCH) = 1.0 (40) krel(HCH) = 1.0 (18) krel(HCH) = 1.0 (42)
(6) Equilibrium trFRET (16) Equilibrium bulk FRET (40) Multiple-turnover cleavage rate (18) Multiple-turnover cleavage rate (42) Single-turnover cleavage rate
HS3H
Pdock =2.9x10-6 (∆∆Gdock = 32 kJ/mol)
∆∆Gdock = 9.6 kJ/mol (6) krel(HCH) = 0.01–0.04 (40) krel(HS9H) = 0.01 (41)
(6) Equilibrium trFRET (40) Multiple-turnover cleavage rate (41) Single-turnover ligation rate
HS6H
Pdock = 8.5x10-2 (∆∆Gdock = 6.1 kJ/mol)
∆∆Gdock = 4.5 kJ/mol (6) ∆∆Gdock = 3.4 kJ/mol (19) [Mg2+]1/2 = 30 mM (16)
(6) Equilibrium trFRET (19) Equilibrium trFRET (16) Equilibrium bulk FRET
HS7H
Pdock = 0.15
krel(HCH) = 0.9–2.1 (40) krel(HS9H) = 1.0 (41)
(40) Multiple-turnover cleavage rate (41) Single-turnover ligation rate
HS9H
Pdock = 0.24
krel(HS9H) = 1.0 (41)
(41) Single-turnover ligation rate
HS10H
Pdock = 0.29
krel(HCH) = 1.1–4.3 (40)
(40) Multiple-turnover cleavage rate
Pdock = 0.45
krel(HCH) = 1.0 (42)
(42) Single-turnover cleavage rate
HCSH-1S1
Pdock = 0.23
krel(HCH) = 1.0 (42)
(42) Single-turnover cleavage rate
HHH
Pdock = 5.4x10-4 (∆∆Gdock = 19 kJ/mol)
∆∆Gdock = 8.2 kJ/mol (6) ∆∆Gdock = 5.1 kJ/mol (19) krel(HCH) = 0.5 (18)
(6) Equilibrium trFRET (19) Equilibrium trFRET (18) Multiple-turnover cleavage rate
HHS2H
Pdock = 0.54 (∆∆Gdock = 1.5 kJ/mol)
∆∆Gdock = 6.0 kJ/mol (*) krel(HCH) = 0.11 (18)
(*) This study (18) Multiple-turnover cleavage rate
HHS4H
Pdock = 0.71
krel(HCH) = 1.5 (18)
(18) Multiple-turnover cleavage rate
H-1SCHS1
5
