Optimization of site-specific RNase H cutting of 1037 nt renilla luciferase mrna. The digestion

Transcription

1 SUPPLEMENTARY FIGURE 1. Optimization of site-specific RNase H cutting of 1037 nt renilla luciferase mrna. The digestion mixture was incubated at 37 C for 0 to 120 minutes. The cutting was nearly complete in minutes. 1

2 SUPPLEMENTARY FIGURE 2. Pre-ligation complex formation between a 40 nt DNA splint (1x molar) and two 20 nt RNA oligonucleotides (1x and 1.2x molar) in different buffer and annealing conditions. The sequences of the DNA splint and RNA oligonucleotides mimick the firefly luciferase mrna sequence at the ligation site defined in Figure 2A. The gel is native PAGE. Lane 1: the DNA splint; lane 2: the two RNA oligonucleotides; lane 3: pre-ligation complex formation by incubation in water at room temperature; lanes 4-10: pre-ligation complex formation by annealing in H 2 O (lane 4), TE (lane 5), TE + 200mM NaCl (lane 6), TE + 75mM LiCl (lane 7), TE + 45% formamide (lane 8), T4 RNA ligase 2 buffer (lane 9), and T4 DNA ligase buffer (lane 10). The pre-ligation complex formation was complete and stable in all tested conditions. 2

3 SUPPLEMENTARY FIGURE 3. Titration of DNA splint molar ratio for the pre-ligation complex formation between 92 nt and 295 nt RNA substrates and a 60 nt DNA splint (upper panel) or a 40 nt DNA splint (lower panel). The 92 nt and 295 nt RNA substrates were at 5:1 (upper panel) or 1:1 (lower panel) molar ratio. Both gels were native PAGE. In both cases, the highest efficiency of pre-ligation complex formation was obtained at around 1 molar ratio of DNA splint. Overall, the extent of preligation complex formation was rather limited for these long RNAs, in contrast to the complete annealing observed for oligonucleotides shown in Supplementary Figure 2. 3

4 SUPPLEMENTARY FIGURE 4. Annealing and ligation of 92 nt and 295 nt RNA fragments using a 60nt DNA splint in different buffer conditions. The RNA fragments and DNA splint were at equimolar ratio. The highest ligation efficiency of approximately 38% was observed for T4 RNA ligase 2 buffer. 4

5 SUPPLEMENTARY FIGURE 5. Cooling speed during annealing does not affect the ligation efficiency of 92 nt and 295 nt RNA fragments, as shown by denaturing PAGE. For annealing, all annealing mixtures were heated to 85 C for 30 seconds, and cooled from 85 C to 4 C with the specified cooling speed. 5

6 SUPPLEMENTARY FIGURE 6. GMP incorporation efficiency during in vitro transcription characteried by XRN-1 digestion. A 295 nt RNA with mono-phosphate group at the 5 end was made by in vitro transcription reaction supplemented with 20 excess of GMP over GTP. XRN-1 (1U/μg) digestion of this RNA (upper gel) is 95% complete after 60 minutes of incubation (lane 4), indicating efficient GMP incorporation at the 5 end. As a control, triphosphorylated RNA (lower gel) is not affected by XRN-1. 6

7 SUPPLEMENTARY FIGURE 7. Ligation of the 1.9 kb firefly luciferase mrna using a 40 nt long DNA splint and different combinations of DNA proximal disruptors. The experiment was identical to Figure 4, except that a 40 nt (vs. 60 nt) long DNA splint was used here for ligation. 7

8 SUPPLEMENTARY FIGURE 8. Quantification (lower right panel) of acrylamide-urea gel (SYBR Green II and Cy5 channels, left panels) for the single-step three-part ligation (upper right panel) for assembling the 1.9 kb firefly luciferase mrna. Additional information is shown in Figure 5 and Table 1. 8

9 SUPPLEMENTARY TABLE 1. Oligonucleotides used for ProDAL of kb-long RNAs. PCR primers for generating DNA templates for in vitro T7 transcription DNA splints used for ligation RNA insert DNA proximal disruptors used for ligation Oligonucleotides used for RNase H cutting Identity and purpose forward for 574 nt firefly fragment reverse for 574 nt firefly fragment forward for 1313 nt firefly fragment reverse for 1313 nt firefly fragment 40 nt splint for firefly * 60 nt splint for firefly 60 nt splint for firefly * 60 nt splint for renilla * 80 nt splint for firefly with 20 nt RNA insert 80 nt splint for firefly with 20 nt Cy5 RNA insert 20 nt Cy5 RNA insert 20 nt RNA insert L1** for firefly R1** for firefly L2** for firefly R2** for firefly L1 for firefly R1 for firefly L2 for firefly R2 for firefly L1 for renilla R1 for renilla L2 for renilla R2 for renilla renilla firefly firefly Sequence CAAGGAATGGTGCATGCTAA CCTTTTTGGAAACGAACACCAC GCCTACCGTGGTTAATACGACTCACTATAGGTTGCAAAAAATTTTG CGAATTCATGCATTTTTTTTTTT GTTCAAAATTTTTTGCAACCCCTTTTTGGAAACGAACACC TTTTTTGCACGTTCAAAATTTTTTGCAACCCCTTTTTGGAAACGAACACCACGGTAGGCT TTAGGCAGACCAGTAGATCCAGAGGAGTTCATGATCAGTGCAATTGTCTTGTCCCTATCG ATAGCTATAATGAAATGCCAAACAAGCACCCCAATCATGGCCGACAAAAATGATCTTCTT TTTTTTGCACGTTCAAAATTTTTTGCAACCAACTTCAACTTAATACCACACCTTTTTGGAAA CGAACACCACGGTAGGCT TTTTTTGCACGTTCAAAATTTTTTGCAACCTTTGTTGTTGTTGAGTTGTGTCCTTTTTGGAAA CGAACACCACGGTAGGCT ph-acacaacu /icy5/caacaacaacaaa ph-ugugguauua AGUUGAAGUU GCCCATACTGTTGAGCAATTCACGTTCATTATAAATGTCGTTCGCGGGCGCAACTGCAAC CGACTGAAATCCCTGGTAATCCGTTTTAGAATCCATGATAATAATTTTTTGGATGATTGG GGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGC TTGTCTTGTCCCTATCGAAGGACTCTGGCACAAAATCGTATTCATTAAAACCGGGAGGTA GCACAAAATCGTATTCATTAAAACCGGGAGGTAGATGAGATGTGACGAACGTGTACATCG ATTGCCAAAAATAGGATCTCTGGCATGCGAGAATCTCACGCAGGCAGTTCTATGAGGCAG CTGGTAATCCGTTTTAGAATCCATGATAATAATTTTTTGGATGATTGGGAGCTTTTTTTG GTGTAGTAAACATTCCAAAACCGTGATGGAATGGAACAACACTTAAAATCGCAGTATCCG AGAAGTTCAAACCATGCAGTAAGATATTTGTAATGATCAAGTAACCTATAAGAACCATTA CATCCCATGATTCAATCACATCTACTACACTTTCAGCGTGAACTATTGCTTTGATCTTAT CTGATTTGCCCATACCAATAAGGTCTGGTATAATACACCGCGCTACTGGCTCAATATGTG TTCTCCAAAACCATTTTTTCTCCTTCTTCAGATTTGATCAACGCAATATCTTCTTCAATA mcmamcmcccaamumcmamu mamamcmcccttmumumumg mgmumumcatgamumcmamg *,, and ligation sites are defined as in Figure 2. ** L1, R1, L2, R2 are defined as in Figure 4A.