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A Synthetic Approach to Stop Codon Scanning Mutagenesis

Lihua Nie, Jason Lavinder, Mohosin Sarkar, Kimberly Stephany and Thomas J. Magliery*

Departments of Chemistry and Biochemistry, The Ohio State University, 100 W. 18th Ave., Columbus, Ohio 43210

*E-mail magliery@chemistry.ohio-state.edu

Supporting Information

Table of Contents Part I: Additional detailed procedures Part II: NMR and mass spectrum of Fmoc-T, Fmoc-TA, Fmoc-TAG, Fmoc-TAG

phosphoramidite and p-Benzoylphenylalanine Part III: Analysis of the relative reactivity of Fmoc-TAG phosphoramidite to

mononucleotide phosphoramidite with HPLC method Part IV: Solid phase DNA library synthesis Part V: The primers used for construction of the pMRH6sup3 and cloning the TAG

mutants. Plasmid maps of pMRH6sup3 with lac and T7 promoter and plasmid map of pSup-Rop-AV-Bpa

Part VI: Gel filtration graph of standards Part VII: Formaldehyde crosslinking and additional UV crosslinking experiments

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Part I: Additional detailed procedures

1H and 31P NMR analysis were performed on 500 and 250 MHz Bruker DRX-500 and

DPX-250. DMSO (d6) was used as solvent for Fmoc-T and CDCl3 were used for Fmoc-TA,

Fmoc-TAG and Fmoc-TAG phosphoramidite. Mass spectra were obtained on Bruker Esquire

LC-MS and Bruker Reflex III MALDI-TOF with positive method. All reactions were monitored

with TLC on glass-backed silica gel 60 F254 sheets (Silicycle). All products were purified by

flash column chromatography using silica gel 60 (Silicycle) with a gradient of CH3OH in CH2Cl2

as elution. The oligonucleotide was synthesized on an Applied Biosystems 392 DNA/RNA

synthesizer.

Fmoc-Cl was from Chempep Inc., DMSO (d6) and CDCl3 were from Aldrich, thymidine,

deoxyadenosine (n-bz) and deoxyguanosine (n-ibu) were from Chemgenes. Triethylamine, 1, 2,

4-Triazole and 1-methylimidazole were from Fluka. 2-chlorophenyl dichlorophosphate, DBU (1,

8-diazabicyclo [5.4.0]-undec-7-ene) and 2-cyanoethyl N, N-diisopropylchlorophosphoramidite

were from Aldrich. Trifluoroacetic acid (TFA) is from Fisher Biotech. Ammonium hydroxide is

from Fisher. Acetonitrile anhydrous is from Pharmco-AAPER. Acetonitrile HPLC grade is from

Fisher. PCR primers were from Sigma. Restriction enzymes and other enzymes for cloning were

from New England Biolabs.

All the reagents and columns used for DNA oligonucleotides synthesis and the

oligonucleotide purification cartridge were from Glen Research.

Crosslinking with UV irradiation was conducted in vivo and in vitro. For the in vivo

crosslinking, 50 mL cell culture was harvested and re-suspended in 1 mL PBS buffer for 30 min

and 2 hour UV irradiation experiments. The cells were placed in a 50 × 15 mm Petri dish on ice,

and a 6 W, 365 nm hand-held UV light was used for the irradiation. The cells were then lysed

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with 0.1 mm glass beads (Biospec) and the protein was purified by Ni-NTA agarose (Qiagen).

For in vitro UV irradiation and formaldehyde crosslinking, 6.7 µM F14Bpa Rop and 10 µM BSA

(if added) were diluted in PBS buffer. The formaldehyde crosslinking experiments were

conducted at 37 oC with 0.9% (v/v) formaldehyde in PBS buffer for 3 hours. The formaldehyde

crosslinking product was precipitated by 4 volumes of cold acetone.

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Part II: NMR and mass spectra of Fmoc-T, Fmoc-TA, Fmoc-TAG, Fmoc-TAG

phosphoramidite and p-Benzoylphenylalanine.

Figure S1. 1H NMR of Fmoc-T, (500 MHz, DMSO, d6): δ 11.3 (1H, s, NH), 7.9 (2H, d, J = 16

Hz, CH on Fmoc), 7.6 (2H, d, J = 7.5 Hz, CH on Fmoc), 7.4 (3H, m, CH on Fmoc and 6-H of

thymidine), 7.3 (2H, m, CH on Fmoc), 6.17 (1H, t, J = 6.5 Hz, 1'-H of thymidine), 5.4 (1H, d, J =

4 Hz, 3'-OH) 4.57 (2H, m, CH2 of Fmoc), 4.31 (1H, m, CH of Fmoc), 4.25 (2H, m, 5'-H of

thymidine), 4.19 (1H, s, 3'-H of thymidine), 3.9 (1H, m, 4'-H of thymidine), 2.1 (2H, m, 2'-H of

thymidine), 1.7 (3H, s, CH3 of thymidine). Peaks at ~3.3 ppm and ~2.5 ppm are from water and

non-deuterated DMSO.

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Figure S2. 13C NMR of Fmoc-T. (500 MHz, DMSO, d6): δ 164.1, 154.3, 150.9, 145.74, 145.70,

141.3, 136.3, 128.2, 127.6, 126.3, 120.7, 110.2, 84.3, 83.9, 70.6, 69.2, 67.95, 46.8, 38.97, 12.6.

Figure S3. Mass spectrum of Fmoc-T. (ESI) m/z: C25H24O7N2, 465.2 [M + H]+, 487.2 [M + Na]+.

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Figure S4. 1H NMR of Fmoc-TA. (500 MHz, CDCl3): δ 11 (1H, NH), 9.9 (1H, NH), 8.75 (1H,

s, 2-H of adenosine), 8.4 (1H, d, J = 20 Hz, 8-H of adenosine), 8.0 (2H, d, J = 7.5 Hz, CH of

Fmoc), 7.7 (2H, d, J = 4.5 Hz, CH on Fmoc), 7.6-7.0 (14H, m, aromatic proton of Fmoc, Bz,

chlorophenyl and 6-H of thymidine), 6.5 (1H, d, J = 10.5 Hz, 1'-H of adenosine), 6.2 (1H, d, J =

8 Hz, 1'-H of thymidine), 5.6 (1H, m, 3'-H of thymidine), 4.7 (1H, m, 3'-H of adenosine), 4.6-4.1

(9H, m, 5'-H, 4'-H of adenosine and thymidine, CH2 and CH of Fmoc), 2.9 (1H, m, 2'-H of

adenosine), 2.55 (2H, m, 2'-H of adenosine and thymidine), 2.1 (1H, m, 2'-H of thymidine), 1.65

(3H, s, CH3 of thymidine).

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Figure S5. 13C NMR of Fmoc-TA (CDCl3, 400 MHz). δ 165.9, 164.19, 164.15, 154.57, 152.39,

151.53, 151.47, 150.7, 150.64, 149.65, 149.61, 146.78, 145.9, 145.83, 142.9, 142.34, 141.27,

141.22, 137.59, 135.09, 133.46, 133.36, 132.92, 130.85, 128.70, 128.22, 128.04, 127.20, 127.15,

126.74, 125.28, 125.21, 124.85, 123.45, 123.40, 121.31, 120.39, 120.15, 111.69, 85.08, 79.15,

70.8, 70.2, 68.57, 67.96, 66.83, 46.66, 39.58, 38.5, 12.47.

Figure S6. 31P NMR for Fmoc-TA. (CDCl3, 250 MHz) δ -7.8 (d).

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Figure S7. Mass spectrum of Fmoc-TA (ESI MS). m/z: C48H43O13N7PCl, 992.4 [M + H]+.

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Figure S8. 1H NMR of Fmoc-TAG. (CDCl3, 500 MHz) δ 12.1 (1H, s, NH of guanosine), 10.9-

10.4 (2H, m, NH of thymidine and guanosine), 10.0 (1H, s, NH of adenosine), 8.75 (1H, s, 2-H

of adenosine), 8.4-8.3 (2H, ss, 8-H of adenosine and guanosine), 8.1-6.8 (aromatic H of Fmoc,

benzoyl and chlorophenyl, 6-H of thymidine), 6.6-6.45 ( 1H, s, 1’-H of adenosine), 6.3-6.1 (2H,

ss, 1’-H of guanosine and thymidine), 5.7-5.5 (1H, s, 3’-H of adenosine), 5.1 (1H, s, 3’-H of

thymidine), 4.7-4.1 (13H, 5’-H, 4’-H of thymidine, adenosine, guanosine, 3’-H of guanosine,

CH2, CH of Fmoc group), 3.4-2.0 (7H, 2’-H of thymidine, adenosine, guanosine and CH of

isopropyl), 1.7 (3H, s, CH3 of thymidine), 1.2 (6H, CH3 of isopropyl).

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Figure S9. 31P NMR of Fmoc-TAG. (CDCl3, 250 MHz) δ -7.31, -7.98.

Figure S10. 13C NMR of Fmoc-TAG (CDCl3, 500 MHz). δ 165.3, 155.63, 154.54, 152.34,

151.44, 150.58, 149.97, 148.04, 145.85, 142.95, 142.62, 141.25, 133.44, 132.69, 130.83, 128.59,

128.2, 127.2, 126.69, 125.2, 124.87, 123.94, 122, 121.33, 120.13, 111.53, 85.23, 84.84, 83.45,

79.33, 71.79, 71.08, 70.22, 69.17, 67.56, 66.76, 58.24, 46.68, 38.23, 37.31, 35.90, 19.44, 19.07,

18.85, 18.41, 18.36, 12.40.

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Figure S11. Mass spectrum of Fmoc-TAG (ESI MS). m/z: C68H64O20N12P2Cl2, 1501 ([M+H]+, 35Cl, 35Cl), 1503 ([M+H]+, 35Cl, 37Cl), 1505 ([M+H]+, 37Cl, 37Cl). 1523 ([M+Na]+

, 35 Cl, 35Cl),

1525 ([M+Na]+, 35Cl, 37Cl), 1527 ([M+Na]+, 37Cl, 37Cl).

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Figure S12. 1H NMR of Fmoc-TAG phosphoramidite (CDCl3, 500 MHz). δ 10.0-9.5 (NH of

thymidine and guanosine), 8.78 (1H, d, J = 10 Hz, 2-H of adenosine), 8.4-8.3 (2H, dd, J = 5 Hz,

7 Hz, 8-H of adenosine and guanosine), 8.0 (2H, d, J = 5 Hz, aromatic H of Fmoc), 7.7 (2H, s,

aromatic H of Fmoc), 7.6-6.9 (9H, m, aromatic H of Fmoc, benzoyl and chlorophenyl, 6-H of

thymidine), 6.6-6.45 (1H, m, 1'-H of adenosine), 6.2 (2H, m, 1'-H of guanosine and thymidine),

5.75-5.65 (1H, s, 3'-H of adenosine), 5.2-5.0 (2H, s, 3'-H of thymidine), 4.7-4.1 (13H, m, 5'-H,

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4'-H of thymidine, adenosine, guanosine, 3'-H of guanosine, CH2, CH of Fmoc group), 4.0-3.6

(4H, m, CH2 of CNCH2CH2), 3.4-2.9 (3H, m, CH of isopropyl), 2.7-2.0 (8H, m, 2'-H of

thymidine, adenosine, guanosine and CH2 of CNCH2), 1.7 (3H, s, CH3 of thymidine), 1.3-1.0

(18H, m, CH3 of isopropyl).

Figure S13. 13C NMR of Fmoc-TAG phosphoramidite (500 MHZ, CDCl3) δ 179.58, 165.5,

173.7, 155.43, 154.52, 150.37, 147.96, 147.46, 145.39, 143.01, 141.2, 139.06, 132.59, 130.8,

130.28, 128.55, 128.2, 127.98, 127.1, 127.0, 126.66, 126.58, 124.87, 124.82, 124.78, 123.16,

121.38, 120.09, 118.08, 111.5, 86.82, 84.95, 84.82, 84.69, 84.61, 79.02, 74.5, 70.2, 69.0, 67.92,

66.80, 57.8, 53.42, 46.7, 43.5, 38.04, 35.6, 24.6, 20.51, 19.5, 18.1, 12.31.

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Figure S14. 31P NMR of Fmoc-TAG phosphoramidite. (CDCl3, 400 MHz) δ 149 (P(III), m), -

7.33, -8.04 (P(V), d)

Figure S15. Mass spectrum of Fmoc-TAG phosphoramidite (TOF MS). m/z: C77H82O21N14P3Cl2, 1700.9 ([M+H]+, 35Cl, 35Cl), 1702.9 ([M+H]+, 35Cl, 37Cl), 1704.9 ([M+H]+, 37Cl, 37Cl). 1722.9 ([M+Na]+

, 35 Cl, 35Cl), 1724.9 ([M+Na]+, 35Cl, 37Cl), 1726.9 ([M+Na]+, 37Cl, 37Cl).

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Figure S16. 1H NMR of p-Benzoylphenylalanine (Bpa). (250 MHz, D2O) δ 7.7-7.2 (m, 9H,

aromatic), 3.79 (t, 1H), 2.90 (q, 1H), 3.15 (q, 1H).

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Figure S17. Mass spectrum of Bpa. (ESI) m/z: 270.3 [M+H]+.

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Part III: Analysis of the relative reactivity of Fmoc-TAG phosphoramidite to DMT-

mononucletide phosphoramidites by HPLC

Fmoc-TAG phosphoramidite at 0.05 M and DMT-mononucleoside at 0.05 M were mixed online

to competitively react with a column which has 40 nmol DMT-adenosine attached to the resin.

The oligonucleotides were then cleaved from the column and the protecting groups were

removed by concentrated ammonium hydroxide at 60 oC. The mixture of the oligonucleotides

was then subjected to HPLC analysis (C18 column, solvent A: H2O with 0.1 M TEAA solution,

pH7; solvent B: CH3CN. Gradient: 0-20% B in 44 minutes). The UV absorption was obtained at

260 nm. The total absorbance of each peak was obtained by integration of the peak area and

indicated on the top of each peak. The relative reactivity of Fmoc-TAG phosphoramidite to each

DMT-mononucleotide phosphoramidite was calculated by using the equation:

%

TAGA

TAGA

XA

XA

A

Fmoc TAGA

ε

ε− =

where ε is the extinction coefficient and X stands for A, T, G, or C.

The extinction coefficient of the chemically synthesized DNA was calculated from the

Applied Biosystems website. In all of these HPLC spectra, retention time (Rt) = 12.73 min is the peak

of A which is presumably from unreacted A on the column.

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Figure S18. HPLC analyses the mixture of TAGA and AA.

Rt = 26.30 min is the peak of AA (260 nm), its absorbance at 260 nm was calculated by integration of the

peak area and indicated as 12194624. Rt = 30.76 min is the peak of TAGA, its absorbance at 260 nm was

2169956. The extinction coefficient for AA and TAGA was calculated to be 27400 and 46700. So, the

molar ratio of TAGA: AA = (2169956/46700): (12194624/27400) = 10.4: 100. And the reactivity of

Fmoc-TAG phosphoramidite compare to DMT-A phosphoramidite is 10.4%.

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Figure S19. HPLC analysis of the mixture of TAGA and GA. GA: Rt = 21.75 min. Absorbance (260 nm) = 13291082. ε = 25200.

TAGA: Rt = 30.69 min. Absorbance (260 nm) = 4490765. ε =46700.

Reactivity Fmoc-TAG/DMT-G = (4490765/46700)/(13291082/25200) = 18.2%.

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Figure S20. HPLC analysis of the mixture of TAGA and CA. CA: Rt = 19.84 min. Absorbance = 11721258. ε = 21200.

TAGA: Rt= 30.36 min. Absorbance = 4061850. ε = 46700.

Reactivity: Fmoc-TAG/DMT-C = (4061850/46700)/(11721258/21200) = 15.7%.

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Figure S21. HPLC analysis of the mixture of TAGA and TA. TA: Rt = 25.17 min. Absorbance = 7942840. ε = 23400.

TAGA: Rt = 30.44 min. Absorbance = 3118832. ε = 46700.

Reactivity: Fmoc-TAG/DMT-T = (3118832/46700)/(7942840/23400) = 19.8%.

Note that the Fig. 4 graph in the manuscript is from the average of three trials with standard

errors shown as error bars (Kaleidagraph 4).

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Part IV: Solid phase DNA library synthesis

The programmed sequence for Rop library:

5’cacacaggaaacacgtatg(a6)c(5c)(a6)a(5a)(c6)a(5g)gaa(a6)a(5a)(a6)

c(5c)gca(c6)t(5t)(a6)a(5c)(a6)t(5g)gcc(c6)g(5c)tag(t6)t(5t)atcag

aagtcagacattaacgc3’

The parenthesis indicates these two nucleotides will be equally imported into the column at the

same time. In this sequence, “5” represents Fmoc-TAG phosphoramidite. “6” indicates DBU will

be sent into the column for deprotection of the Fmoc group.

Here is an example of the program: (“y”: yes, “-”: No)

Step function time (s) A G C T 5 6 7 8 69 14(TCA) to column 6 y y y y y y - - 70 Trityl flush 5 y y y y y y - - 71 10 (DBU) to column 6 - - - - - y - - 72 Trityl flush 5 y y y y y y - -

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Part V: The primers used for construction of the pMRH6sup3 and cloning the TAG

mutants. And the plasmid maps of pMRH6sup3 with lac and T7 promoter

Primers used for construction of pMRH6sup3 Set1-5 TAAGATGCGCCCCGCATTGGGGCTATAGCTCAGCTG Set1-3 TACACGGCGGAGACTACTTGGTGGAGCTAAGCGGGATC Set2-5 CAAGTAGTCTCCGCCGTGTAGCAAGAAATTGAGAAGTGGGGCTATAGCTCAGCTG Set2-3 TAAAACCTCTTCAAATTTGCCGTGCAAATTTGGTGGAGCTAAGCGGGATC Set3-5 GCAAATTTGAAGAGGTTTTAACTACATGTTATGGGGCTATAGCTCAGCTG Set3-3 TCGTTGAGCAGGCTTTTCGAATTTGGTGGAGCTAAGCGGGATC Amp1-5 AAGGCATTTTGCTATTAAGGGATTGACGAGGGCGTATCTGCGCAGTAAGATGCGCCCC GCATTG Amp1-3 AATAATGGCACCATGCAAAAAAGCCTGCTCGTTGAGCAGGCTTTTCG Amp2-5 AATAATGGTGCCGTGCACGGCTAACTAAGCGGCCTGCTGACTTTCTCGCCGATCAAAA GGCATTTTGCTATTAAGG Amp2-3 CGAGATGCAAAAAAGCCTGCTCGTTGAGCAGGCTTTTCG tRNApoly5 AATCTGCAGGTGCACGGCTAACTAAGCGGCCTG tRNApoly3 AATAGATCTCGAGATGCAAAAAAGCCTGCTCGTTG Final sequence of the polycistron with three EcalaWtRNACUA genes (each in red) AATCTGCAGGTGCACGGCTAACTAAGCGGCCTGCTGACTTTCTCGCCGATCAAAAGGC ATTTTGCTATTAAGGGATTGACGAGGGCGTATCTGCGCAGTAAGATGCGCCCCGCATTG GGGCTATAGCTCAGCTGGGAGAGCGCTTGCATGGCATGCAAGAGGTCAGCGGTTCGAT CCCGCTTAGCTCCACCAAGTAGTCTCCGCCGTGTAGCAAGAAATTGAGAAGTGGGGCT ATAGCTCAGCTGGGAGAGCGCTTGCATGGCATGCAAGAGGTCAGCGGTTCGATCCCGC TTAGCTCCACCAAATTTGCACGGCAAATTTGAAGAGGTTTTAACTACATGTTATGGGGC TATAGCTCAGCTGGGAGAGCGCTTGCATGGCATGCAAGAGGTCAGCGGTTCGATCCCG CTTAGCTCCACCAAATTCGAAAAGCCTGCTCAACGAGCAGGCTTTTTTGCATCTCGAGA TCTATT

Primers used for construction of TAG containing mutants and site specific alanine mutagenesis TAG-scanned oligonucleotide: CACACAGGAAACACGTATGACCAAACAGGAAAAAACCGCACTTAACATGGCCCGCTTT ATCAGAAGTCAGACATTAACGC TAGscanRev: GTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATATCTGCCTGTTCATCCGCGT CCAGCTCGTTGAGTTTCTCTAAAAGCGTTAATGTCTGACTTCTG TAGscan5: CACACAGGAAACACGTATG TAGscan3: AATAATGGCACCTCAATGATGATGATGGTGATGTCCTCCGAGGTTTTCACCGTCATCTC CGAAACGCGCGAGGCAAGAACGGTAAAGCTCATCAGCGTGG RopAla2: GCGAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG RopAla3: ACCGCGCAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG RopAla6: ACCAAACAGGAAGCGACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG RopAla10: ACCAAACAGGAAAAAACCGCCCTTGCGATGGCCCGCTTTATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG RopAla11: ACCAAACAGGAAAAAACCGCCCTTAACGCGGCCCGCTTTATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG RopAla14 : ACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCGCGATCAGAAGCCAGACATTAA CGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG

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SM-wtROP3: GCCATCGTCACCAAAGCGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGA AGCGATTCACAGATATCTGCCTGTTCATCCGCGTCCAGCTCG pMRsup3Ala0-5 : AATATTACACGTATGACCAAACAGGAAAAAACC pMRsup3Ala2-5: AATATTACACGTATGGCGAAACAGGAAAAAACC pMRsup3Ala3-5: AATATTACACGTATGACCGCGCAGGAAAAAACC pMRsup3Ala6-5: AATATTACACGTATGACCAAACAGGAAGCGACC SM-pAC3: CAGTGAGGCACCTCAGAGGTTTTCGCCATCGTCACCAAAGCG LacTAG5: AATGGTGGTCACCTAATACGAC LacTAG3: TAATCTAGACGTTAAGGGATTTTGGTCATGAG 5TAG2T7: GCTCGTATGTTGTGTGGAATTG pMRH6TEVLeu-3: TAATAAGGATCCTCAGAGGTTTTCACCGTCATCACCGAAACG Afl3cass5: GTAGTCTCCGCCGTGTAGCAAG Afl3cass3: CGTGGCCAATATGGACAACTTCTTC

Plasmid maps

Figure S22. The constructed pMRH6sup3 vector with 3 sets of tRNA under proK promoter

control and Rop variants under lac and T7 promoter.

Figure S23. The plasmid map of pSup-Rop-AV-F14Bpa.

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Part VI: Gel filtration graph of standards.

Figure S24. FPLC of standard proteins and the plot of logM to volume (inset).

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Part VII: Formaldehyde crosslinking experiments

Figure S25. Formaldehyde crosslinking experiments. The crosslinking experiments were done at

37oC with 0.9% formaldehyde in PBS buffer for 3 hours.

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Figure S26. UV crosslinking with F14Bpa Rop. Lane 1: wild type Rop without UV irradiation;

lane 2: F14Bpa Rop without UV irradiation. (The small amount of dimer and trimer results from

room light during the protein expression and purification procedure.); lane 3: F14Bpa Rop

protein was irradiated by 365 nm UV light for 30 min; lane 4: F14Bpa Rop protein was

irradiated for 120 min; lane 5: protein marker.