Supporting Information

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1 Translation of DNA into Synthetic N-Acyloxazolidines Xiaoyu Li, Zev. J. Gartner, Brian N. Tse and David R. Liu* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts Supporting Information DNA synthesis and analysis DNA oligonucleotides were synthesized on a PerSeptive Biosystems Expedite 8090 DNA synthesizer using standard phosphoramidite protocols and purified by reverse phase HPLC with a triethylammonium acetate (TEAA)/CH 3 CN gradient. Amino-modifier C6-dT (Glen Research) was used for the preparation of T template. The 5 -amino modified oligonucleotides were prepared by standard automated DNA synthesis using the 5 -amino-modifier 5 phosphoramidite from Glen Research. The 3'-thiol modified oligonucleotides were prepared by standard automated DNA synthesis using the 3 -thiol-modifier C3 S-S CPG from Glen Research); the 3 - amine modified oligonucleotides were prepared with the same protocol but with the 3 -aminomodifier C7 CPG modifier from Glen Research. Oligonucleotides were quantitated by UV and by denaturing polyacrylamide gel electrophoresis (PAGE) after staining with ethidium bromide following by UV transillumination. Quantitation of DNA after was performed with a Stratagene Eagle Eye II densitometer. Reaction yields were quantitated by denaturing polyacrylamide gel electrophoresis followed by ethidium bromide staining, UV visualization, and densitometry of product and template starting material bands. Yield calculations assumed that templates and products in denaturing gels stained with comparable intensity per base; for those cases in which products are partially double-stranded during quantitation, changes in staining intensity may result in higher apparent yields. DNA functionalization Functionalization of oligonucleotides using a sulfone linker (3, 7). The corresponding amino acid (DL-β-furylserine for 3 and L-serine for 7) was dissolved in 50 µl of 300 mm NaOH solution stoichiometrically then treated with 150 µl of 100 mm bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES, Pierce) in DMF. From this mixture, 40 µl was added to a 90 µl solution containing up to 150 µg of 3 - or 5 - amino modified DNA dissolved in 200 mm ph 7.2 sodium phosphate buffer. The reaction was maintained at 25 C for 1

2 2 h. The products were purified by gel filtration and reverse-phase HPLC and characterized by denaturing PAGE and MALDI-TOF mass spectrometry. Functionalization of oligonucleotides using a diol linker (10). N-FMOC-1,1 - ethylenediamine hydrochloride was dissolved in 50 µl of 300 mm NaOH solution stoichiometrically before mixing with 150 µl of 100 mm disuccinimidyl tartarate linker (DST, Pierce) solution in DMF. From this mixture, 40 µl was added to a 90 µl solution containing up to 150 µg 3 - or 5 - amino modified DNA dissolved in 200 mm ph 7.2 sodium phosphate buffer. The reaction was maintained at room temperature for 2 h. The intermediate product of N-Fmoc-1,1 -ethylenediamino-dst-modified DNA was purified by reverse-phase HPLC and dissolved in 300 µl of 100 mm NaOH to remove the FMOC group. The deprotection reaction was maintained at 25 C for 1 h500 µl of 3 M NaOAc buffer (ph 4.0). The quenched reaction was desalted by gel filtration the desired 1,1 -ethylenediamino-dstmodified DNA was purified by reverse-phase HPLC. The 1,1 -ethylenediamino-dst-modified DNA was dissolved in 90 µl of 200 mm phosphate buffer (ph 7.2), then combined with 30 µl of a DMF solution containing 30 mg/ml of the NHS ester of p-formylbenzoic acid. The reaction was maintained at 25 C for 1.5 h, and the final modified DNA (10) with a diol linker was purified by reverse-phase HPLC and further characterized by denaturing PAGE and MALDI-TOF mass spectrometry. Functionalization of oligonucleotides using a thioester linker (5). A solution of 10 µl of 3 - thiol modified DNA solution was mixed with 10 µl of 200 mm DTT and 20 µl of 2M TAPS (ph 8.0) to reduce the disulfide bond. The cleavage reaction was maintained at 25 C for 30 min and purified by gel filtration to remove excess DTT and buffer salts. The DNA-containing fraction (1 ml) was lyophilized. The dried intermediate product was resuspended in 90 µl of 200 mm sodium phosphate buffer and mixed with 40 µl of 30 mg/ml p-formylbenzoic acid NHS-ester in DMF. The reaction was maintained at 25 C for 2 h. The product was purified by gel filtration and reversephase HPLC, and further characterized by MALDI-TOF mass spectrometry. Functionalization of oligonucleotides using a phosphine linker (11). A 3 -amino modified DNA was prepared by standard automated DNA synthesis but was not cleaved from CPG beads. p-diphenylphosphinobenzoic acid (300 mg) was dissolved in 3.5 ml dry DMF together with 192 2

3 mg EDC and 360 µl DIPEA. The mixture was maintained under 25 C for 1 h. A 1 ml aliquot of the crude reaction mixture was added to the 3 -amino CPG beads prepared above. The resulting suspension was agitated at 37 ºC for 12 h, filtered, washed (3 x 1.5 ml DMF and 3 x 1.5 ml CH 3 CN), and dried under N 2. The 3 -phosphine-modified DNA was cleaved from the dried CPG beads with 500 µl of 1:1 ammonium hydroxide:methyl amine (AMA) together with 1.2 mg tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl, Aldrich) to prevent phosphine oxidation. The final modified DNA was purified by reverse-phase HPLC and further characterized by MALDI-TOF mass spectrometry. A solution of 20 µl of 1 M serine in 1M NaOH was combined with 12.5 µl of 20 mg/ml N- succinimidyl iodoacetate (SIA, Pierce) in DMF for 10 min before being combined with 90 µl of solution containing up to 150 µg 3 -diphenylphosphine modified DNA dissolved in 200 mm of ph 7.2 sodium phosphate buffer. The reaction was maintained at 25 C and the product was purified by reverse-phase HPLC and further characterized by MALDI-TOF mass spectrometry. DNA-templated reactions DNA-templated synthesis of 4 (Figure 1). The DNA-templated amine acylation reaction was typically performed on a 1 nmol scale. The concentration of T architecture template 2 was 60 nm and the concentration of reagent 3 was 120 nm. The reaction was performed in 100 mm MOPS buffer (ph 7.5) containing 1 M NaCl and 50 mm 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMT-MM, ACROS Chemicals). The reaction was maintained at 37 ºC for 12 h. After the reaction, the crude mixture was incubated directly with streptavidinlinked magnetic beads (Roche, 75 µl beads per 100 pmol biotinylated oligonucleotides). The biotin-streptavidin incubation was agitated at 25 C for 1 h. The beads were then separated on a magnet rack for 1 min. After removing the supernatant, the pellet was washed with denaturing buffer (5 x 400 µl 4 M guanidinium HCl and 1 x 400 µl ddh 2 O, 30 min per wash) to remove species bound to beads purely through Watson-Crick interactions. The washed beads were treated with 600 µl cleavage buffer containing 100 mm CAPS (ph = 11.8) and 20 mm β-mercaptoethanol at 37 C for 1 h under agitation to cleave the sulfone linker. The supernatant from the cleavage, containing product 4, was purified by ethanol precipitation and quantified by UV spectrometry and denaturing PAGE. MALDI-TOF mass spectrometry analysis was consisted with the expected structure (expected mass = ; observed mass = ±11, Figure S1). 3

4 Figure S1: MALDI-TOF analysis of purified 4. Data was collected using an internal oligonucleotide standard and negative ion mode (M-H) -. Expected mass = ; observed mass = ±11. DNA-templated synthesis of 1a (Figure 2). The DNA-templated oxazolidine formation reaction contained 60 nm of template 4 and 120 nm of reagent 5 at 37 C with 1 M NaCl and 100 mm MOPS (ph 7.5). After 6 h, oxazolidine N-acylation reagent 7 was added (final concentration 60 nm) directly into the reaction mixture together with 50 mm DMT-MM. The added volume of 7 was less than 0.5% of the total reaction volume. The resulting reaction was maintained at 37 C for overnight. The crude reaction mixture containing desired 8 and other undesired species (4, 5, 6, 7 and 9) from above was directly incubated with magnetic streptavidin-linked beads using the same protocol as in the synthesis of 4. After removing the supernatant, denaturing washes with 5 x 400 µl of 4M guanadinium HCl and 1 x 400 µl ddh 2 O removed non-biotinylated 4, 7 and 9. The beads were treated with 600 µl of 500 mm NaOAc buffer (ph 3.5) at 25 C for 1h to hydrolyze N-unacylated oxazolidine 6. Denaturing PAGE was used to monitor hydrolysis efficiency (Fig. 2, lane D to E). The resulting beads, which contained only 5 and 8, were washed with an additional 400 µl ddh 2 O. The thioester and sulfone linkers in 8 were cleaved by suspending the beads in 100 mm CAPS buffer at ph 10.0 containing 10 mm β-mercaptoethanol (total volume 600 µl) at 37 C for 1 h under agitation. The non-bead bound species now contained desired product 1a and the 10-mer contaminating oligonucleotide from the sulfone linker cleavage (Fig. 2, lane E to F). Undesired biotinylated 5 remained on the beads. Denaturing PAGE purification of the supernatant provided 1a in 3-6% overall yield from 2. The final product was further characterized by MALDI-TOF mass spectrometry (Figure S2). 4

5 Figure S2: MALDI-TOF analysis of purified 1a. Data was collected using an internal oligonucleotide standard and negative ion mode (M-H) -. Expected mass = , observed mass = ±11. DNA-templated synthesis of 1b (Figure 3). DNA-templated oxazolidine formation was performed using 60 nm 4 and 120 nm 10 at 37 C with 1 M NaCl and 100 mm MOPS (ph =7.5) to form the N-unacylated oxazolidine intermediate 12. After 6 h, acylating reagent 11 was added (final concentration 60 nm) directly to the reaction mixture together with 50 mm DMT-MM. The added volume of 11 was less than 0.5% of the total reaction volume. The reaction was maintained at 37 C for 12 h. The crude reaction mixture containing desired 13 and other undesired species (4, 10, 11, 12 and 14) was combined with magnetic streptavidin-linked beads using the protocol described above. After removing the supernatant, denaturing washes (5 x 400 µl of 4 M guanadinium HCl and 1 x 400 µl ddh 2 O) removed non-biotinylated 4, 10 and 12. The beads were treated with 600 µl of 50 mm NaIO 4 solution in 500 mm NaOAc buffer at ph 4.0 for 2 h to cleave the diol linker and convert 13 to 15. After the cleavage buffer was removed, the beads were washed with 2 x 400 µl 3 M guanadinium HCl in 500 mm NaOAc (ph 4.0) and 1 x 400 µl ddh 2 O to remove the cleaved DNA fragment from 13. Denaturing PAGE was used to monitor diol cleavage efficiency (Fig. 3, lanes D-F). Following the diol linker cleavage, the beads were treated with 600 µl of 500 mm TAPS (ph = 8.5) for 2 h to initiate Wittig macrocyclization. Cyclization converted 15 to the desired product 1b and also cleaves 1b from the streptavidin-linked beads. Desired product 1b was further purified by ethanol precipitation and further characterized by MALDI-TOF mass spectrometry (Figure S3). Undesired biotinylated 11 and 14 remained on the CPG. 5

6 Figure S3: MALDI-TOF analysis of purified 1b. Data was collected using an internal oligonucleotide standard and negative ion mode (M-H) -. Expected mass = , observed mass = ±11. DNA oligonucleotide sequences and modifications T-template 2: 5 -TGA CCA CAC CTT T*GG GTA CGA ACG CGA CTC GGG AT-3. T* indicates the C6-amino-modified dt. Reagent for 1 st reaction (Figure 1, 3): 5 -Biotin-CONH-(CH 2 ) 6 -OPO - 3 -TCC CGA GTC GGT ACC-CH 2 -CH(CH 2 OH)-(CH 2 ) 4 NH 2. Reagent for 2 nd reaction (Figure 2, 5): 5 -Biotin-CONH-(CH 2 ) 6 -OPO - 3 -CGT TCG ATC C- OPO - 3 -(CH 2 CH 2 O) 3 -PO - 3 -(CH 2 ) 2 -SR. R- is the corresponding aldehyde shown in Figure 2. Reagent for 2 nd reaction (Figure 3, 10): 5 -CGT TCG ATC C-OPO - 3 -CH 2 -CH(CH 2 OH)- (CH 2 ) 4 NHCO-R. R- is the diol-linked aldehyde group shown in Figure 3. Reagent for 3 rd reaction (Figure 2, 7): 5 -AGG TGT GGT C-OPO - 3 -CH 2 -CH(CH 2 OH)- (CH 2 ) 4 NHR. R- is the BSOCOES linked serine group shown in Figure 2. Reagent for 3 rd reaction (Figure 3, 11): 5 -Biotin-CONH-(CH 2 ) 6 -OPO - 3 -AGG TGT GGT C- OPO - 3 -CH 2 -CH(CH 2 OH)-(CH 2 ) 4 NHR. R- is the phosphine-linked serine group shown in Figure 3. 6