Supplementary Information for:
A Transition State Analog for an
RNA-editing Reaction
Brittany L. Haudenschild, Olena Maydanovych, Eduardo A. Veliz,
Mark R. Macbeth, Brenda L. Bass and Peter A. Beal*
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
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Figure S1. 1 H-NMR for 2',3',5'-tri-O-acetyl 6-bromo-8azanebularine (2).
Figure S2.
13
C-NMR for 2',3',5'-tri-O-acetyl 6-bromo-8azanebularine (2).
Figure S3. 1 H-NMR for 5'-O-(4,4'-dimethoxyt rityl)-8-azanebularine (5).
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Figure S4. 13 C-NMR for 5'-O-(4,4'-dimethoxytrityl)-8-azanebularine (5).
Figure S5. 1 H-NMR for 5'-O-(4,4'-dimethoxytrityl)-2'-O-(t-butyldimethylsilyl)-8azanebularine (6).
Figure S6. 13 C-NMR for 5'-O-(4,4'-dimethoxytrityl)-2'-O-(t-butyldimethylsilyl)-8azanebularine (6).
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31
Figure S7.
P-NMR for 5'-O-(4,4'-dimethoxytrityl)-3'-O-[(2-cyanoethoxy)(N,Ndiisopropylamino)phosphino]-2'-O-(t-butyldimethylsilyl)-8azanebularine (7).
928.30
5'-A8azaNG-3'
Figure S8. MALDI mass spectrometry analysis of the 5'-A8azaNG-3', after deprotection
with Et3 N•3HF giving the predicted mass of the intact trimer. (Positive ion mode.)
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917.14
5'-AXG-3'
Figure S9. MALDI mass spectrometry analysis of the 5'-A8azaNG-3', after deprotection
with TBAF/THF, 5'-AXG-3', where X is proposed to be a 5-amino-1,2,3-triazole-4carbaldehyde ribosyl residue. (Negative ion mode.)
A
B
ß complex
ß complex
ß free RNA
ß free RNA
Figure S10. (A) Quantitative gel mobility shift analysis of R-D binding to RNA
containing 8-azanebularine at the R/G site. (Top) Storage phosphor autoradiogram of
native polyacrylamide gel separating free from bound RNA. (Bottom) Plot of fraction
bound versus concentration of R-D. Data were fit to the equation: fraction
bound=A*[protein]/([protein] + KD) where KD is the fitted dissociation constant and A is
the fitted maximum fraction RNA bound at that dissociation constant. (B) Analysis of RD binding to RNA containing 8-azanebularine at the A24 site.
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A
B
ß complex
ß complex
ß free RNA
ß free RNA
Figure S11. (A) Analysis of ADAR2 full binding to RNA containing 8-azanebularine at
the R/G site. (B) Analysis of ADAR2 full binding to RNA containing 8-azanebularine at
the A24 site.
ß complex
ß free RNA
Figure S12. Analysis of R-DE396A binding to RNA containing 8-azanebularine at the R/G
site.
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