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ISMB読み会2016

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August 17, 2016

 ISMB読み会2016

RNAiFold2T: Constraint programming design of thermo-IRES switches

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carushi

August 17, 2016
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  1. Science, 2016 Jan. • RNA loop structure, m6A, IRES •

    10%ͷmRNA͕capඇґଘͷ຋༁Λड͚Δ • 5’UTR and 3’UTRͲͪΒʹ΋ΤϯϦον
  2. RNA folding • ഑ྻ→ߏ଄ • ૬ิతͳԘجͷpairingʹΑΓ҆ఆԽ • A-U, G-C, G-U

    • ΔG of GC/CG stacking is • 3.4kcal/mol - ATP hydrolysis 7kcal/mol • ࠷҆ఆʢMFEʣߏ଄Λ༧ଌ Hairpin Internal MulV A Exterior Bulge …((…))… Example
  3. RNA “inverse” folding • ߏ଄ → ഑ྻ • ಛఆͷMFEߏ଄ΛऔΔ഑ྻΛσβΠϯ͢Δ •

    RNA߹੒ͷ෼໺Ͱॏཁ • λʔήοτɿ਺े͔Β਺ඦnt ఔ౓ͷΞϓλϚʔɺϦϘεΠον • φΠʔϒʹ΍Δͱ͍͍ͩͨܭࢉ͸ܹॏ • ഑ྻΛ࡞Δ → MFEߏ଄༧ଌ O(N^3) → ഑ྻΛ࡞Δ…ͷ܁Γฦ͠ • γϡʔυϊοτҎ্ͷෳࡶͳߏ଄ͷMFE͸NP׬શͳͷͰେମ͸ແࢹ
  4. طଘιϑτ΢ΣΞ Թ౓ґଘͰ̎ߏ଄ΛऔΔ • Switch Design (Flamm 2001) - adapVve walk

    • Frnakenstein (Lyngso 2012) - geneVc algorithm • ͲͪΒ΋֘౰͢ΔதͰ୳ࡧΛ͠ ͳ͕Β࠷ద഑ྻΛҰͭग़ྗ͢Δ ॳΊͯݫີղͷྻڍ͕ՄೳͳΞϧΰϦ ζϜΛ࡞ͬͨ Inverse folding • RNAinverse • RNA-SSD • INFO-RNA • MODENA (made in Japan!) • NUPACK-DESIGN (Caltech) • Inv (China) • RNAifold
  5. ΞϧΰϦζϜ֓ཁ • RNAifoldΛϕʔεͷ੍໿෇͖ͷInverse folding • ෳ਺ߏ଄ʹର࣮ͯ͠ߦ • ೋ࣍ߏ଄Λ໦ߏ଄ʹ෼ղ • ϊʔυؒͷoverlapɾinclusion

    • ؔ࿈͢Δϊʔυ͸ಉ࣌ʹ࠷దԽ • ϊʔυͷ୳ࡧॱংΛ֬ఆ ਺ࣈٯʁ ࣮૷ɿC++&OR-tools (CP search engine) ϧʔϓ಺͸͓ͦΒ͘ϚϧνεϨου
  6. 1,2: bp and up 0: bp and bp 4,6: bp

    and paired i 5,6: bp and paired j 7: closing bp
  7. RNAifoldͷઓུ ……((……))…………….((((…))).)…… ໨తͷߏ଄ ᶃಠཱͷϊʔυʹ෼ղ NNGNNNNNNNN ↓ AAGAAAAAACUA ↓ ᶄ഑ྻΛ੍໿ʹैͬͯຒΊΔ ԘجରΛ૊Ή৔ॴ͸Ԙجରͷ

    ϖΞͰɺ૊·ͳ͍৔ॴ͸೚ҙ ͷԘجͰNΛຒΊͯΈΔ .((……)). ᶅRNAfoldͰ໨తͷߏ଄ͱ ߹͏͜ͱΛ֬ೝ ※গͳ͘ͱ΋͜ͷ෦෼഑ྻ͸໨తͷߏ଄ͱҰக͍ͯ͠Δඞཁ͕͋Δ unpairdͷԘج͕पғʹ௥Ճ͞ΕΔ͜ͱͰҧ͏MFEߏ଄ΛऔΓ͏Δ
  8. • ׬੒ͨ͠΋ͷ͔ΒMFEߏ଄Λ νΣοΫ͢Δʢ30℃→10℃ʣ ޻෉ʢHeurisVcʣ • Ԙجର͸҆ఆతͳGC͔ΒೖΕͯ ΈΔ • ยํͰ͸૊·ͳ͍΋ͷ͸ٯʹෆ ҆ఆͳ΋ͷ͔Β

    • ϧʔϓྖҬ͸AU͔ΒೖΕͯΈΔ • ͨͩ͠ϥϯμϜม਺Ͱॱংʹ͸ ༳Β͗ Թ౓ґଘͰ໨తߏ଄͕̎ͭ͋Δ৔߹
  9. ݁Ռɹthermo-IRESͷσβΠϯ 42℃ʢ׆ੑߴ͍ʣ 30℃ʢ׆ੑ௿͍ʣ Experimentally validated! • RNAifold2TͰީิΛڍ͛Δ • ҎԼͷબ୒ج४ͰD1,D2ΛηϨΫτ •

    (a)(b)ͷߏ଄ΛͱΔʢP > 0.2ʣ • ҙਤ͠ͳ͍ߏ଄ͷ֬཰ͷ̎ഒҎ্ • ௚ۙͷυϝΠϯ4ͷߏ଄Λෆ҆ఆԽͤ͞ ͳ͍ Activity 42℃/30℃ WT D1 D2 NC Start codon Py tract • FMDV IRES domain 5 • IRESͷCapඇґଘͳ຋༁͸42℃ͷߏ ଄ͷํ͕Α͘ಇ͘
  10. IRES׆ੑͷ࣮ݧతݕূํ๏ • Luc/CAT • 35S labelled Luciferase polypepVde • chloramphenicol

    acetyl transferase polypepVde • SDS-PAGE → DensitometerͰൃݱྔͷൺΛऔΔ CAT 5’UTR Luciferase Cap
  11. ݁Ռɹ༧ଌ݁Ռൺֱ λ phage thermoregulator RF01804 Relative frequency of the cost

    function of SD→௿͋͘Δඞཁ͸ͳ͍ʁ Black : RNAifoldͷશղ୳ࡧ Λ௕࣌ؒߦͬͨ΋ͷ
  12. Cost function Flamm, et al. RNA. 2001. G: partition function

    →-RTlnp(Ω1)-RTlnp(Ω2) →ͨͩ݁͠Ռ1Ͱ࢖͍ͬͯͨ બ୒ج४ʹྨࣅ
  13. ίϝϯτ • ΞϧΰϦζϜͱͯ͠͸طଘͷ΋ͷʹ৽ͨͳ੍໿Λ௥Ճͨ͠΋ͷ • χον͚ͩͲݫີղ͑Β͍→طଘख๏ͷίετؔ਺͸࣮͸Πέͯͳ͍ͷͰ͸ʁ • ࣮༻తʹ͸༷ʑͳheurisVc͕ඞཁ • MFEʹ͔͠ʢ༗ޮʹʣ࢖͑ͳ͍ɺߏ଄ܭࢉͷ࠶ར༻͸0 •

    ௕͍΋ͷʹରͯͨ͘͠͞ΜͷީิΛग़ͤΔͱ͜Ζʹ༏Ґੑ • ͋ͱ͸ؤு࣮ͬͯݧͱ͍͏ํ޲ੑ • ੜ෺ֶతʹ໘ന͍τϐοΫʹ࢖͑Δʢͨʣ఺͕ධՁ͞Εͨͱ͍͏ҹ৅