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ISMB読み会2016
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carushi
August 17, 2016
Science
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ISMB読み会2016
RNAiFold2T: Constraint programming design of thermo-IRES switches
carushi
August 17, 2016
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Transcript
*4.#ಡΈձ http://www.slideshare.net/carushi
• ԹʹԠͯ͡ೋ࣍ߏ͕มԽ͠ɺRNAͷ࣋ͭػೳΛௐઅ͢ΔRNA termometersͷ ྻΛσβΠϯ͢ΔΞϧΰϦζϜΛ։ൃ • ࢬמΓˍ୳ࡧΛ܁Γฦ͢ݫີղʹՃ͑ͯlarge neighbourhood search algorithmʹΑ ΔᩦཉղΛಘΔ
• 42℃→30℃Ͱ50%׆ੑ͕མͪΔThermo-IRESεΠονΛ࣮ݧతʹ֬ೝ
• ࣮ݧεϖΠϯͷάϧʔϓͱڞಉ • IRESͷߏղੳ • ͪ͜ΒͷจԹґଘੑͰͳ͘ɺೋ࣍ߏΛ յ͢ྨࣅྻΛσβΠϯ • picornavirusͷdomain 5ʹ͓͍ͯϔΞϐϯϧʔϓͱ
ϙϦϐϦϛδϯτϥΫτͷ࿐ग़͕IRESͷ׆ੑʹॏ ཁͰ͋Δ͜ͱΛௐͨ
Science, 2016 Jan. • RNA loop structure, m6A, IRES •
10%ͷmRNA͕capඇґଘͷ༁Λड͚Δ • 5’UTR and 3’UTRͲͪΒʹΤϯϦον
RNA folding • ྻ→ߏ • ૬ิతͳԘجͷpairingʹΑΓ҆ఆԽ • A-U, G-C, G-U
• ΔG of GC/CG stacking is • 3.4kcal/mol - ATP hydrolysis 7kcal/mol • ࠷҆ఆʢMFEʣߏΛ༧ଌ Hairpin Internal MulV A Exterior Bulge …((…))… Example
RNA “inverse” folding • ߏ → ྻ • ಛఆͷMFEߏΛऔΔྻΛσβΠϯ͢Δ •
RNA߹ͷͰॏཁ • λʔήοτɿे͔Βඦnt ఔͷΞϓλϚʔɺϦϘεΠον • φΠʔϒʹΔͱ͍͍ͩͨܭࢉܹॏ • ྻΛ࡞Δ → MFEߏ༧ଌ O(N^3) → ྻΛ࡞Δ…ͷ܁Γฦ͠ • γϡʔυϊοτҎ্ͷෳࡶͳߏͷMFENPશͳͷͰେମແࢹ
طଘιϑτΣΞ ԹґଘͰ̎ߏΛऔΔ • Switch Design (Flamm 2001) - adapVve walk
• Frnakenstein (Lyngso 2012) - geneVc algorithm • ͲͪΒ֘͢ΔதͰ୳ࡧΛ͠ ͳ͕Β࠷దྻΛҰͭग़ྗ͢Δ ॳΊͯݫີղͷྻڍ͕ՄೳͳΞϧΰϦ ζϜΛ࡞ͬͨ Inverse folding • RNAinverse • RNA-SSD • INFO-RNA • MODENA (made in Japan!) • NUPACK-DESIGN (Caltech) • Inv (China) • RNAifold
ΞϧΰϦζϜ֓ཁ • RNAifoldΛϕʔεͷ੍͖ͷInverse folding • ෳߏʹର࣮ͯ͠ߦ • ೋ࣍ߏΛߏʹղ • ϊʔυؒͷoverlapɾinclusion
• ؔ࿈͢Δϊʔυಉ࣌ʹ࠷దԽ • ϊʔυͷ୳ࡧॱংΛ֬ఆ ࣈٯʁ ࣮ɿC++&OR-tools (CP search engine) ϧʔϓ͓ͦΒ͘ϚϧνεϨου
1,2: bp and up 0: bp and bp 4,6: bp
and paired i 5,6: bp and paired j 7: closing bp
RNAifoldͷઓུ ……((……))…………….((((…))).)…… తͷߏ ᶃಠཱͷϊʔυʹղ NNGNNNNNNNN ↓ AAGAAAAAACUA ↓ ᶄྻΛ੍ʹैͬͯຒΊΔ ԘجରΛΉॴԘجରͷ
ϖΞͰɺ·ͳ͍ॴҙ ͷԘجͰNΛຒΊͯΈΔ .((……)). ᶅRNAfoldͰతͷߏͱ ߹͏͜ͱΛ֬ೝ ※গͳ͘ͱ͜ͷ෦ྻతͷߏͱҰக͍ͯ͠Δඞཁ͕͋Δ unpairdͷԘج͕पғʹՃ͞ΕΔ͜ͱͰҧ͏MFEߏΛऔΓ͏Δ
……((……))…………….((((…))).)…… ᶆ֤෦ߏ͔ΒಘΒΕͨྻΛΈ߹Θͤͯ࠷ऴతʹతͷߏͱ߹͏͔֬ೝ RNAifoldͷઓུ AAGAAAAAACUA ACGGGAAACCCAGA ……((……))…………….((((…))).)…… + Target MFE ෆҰகͷ߹
• ਖ਼ղ෦ͷBpΛ֬తʹݻఆ͠࠶୳ࡧ (LNS) • શύλʔϯࢼ͢ (CP)
ԹґଘͰతߏ͕̎ͭ͋Δ߹ 30℃ͷԘجର()෦ΛຒΊΔ 30℃ͷϧʔϓ.෦ΛຒΊΔ 30℃ͷڥք෦ΛຒΊΔ ಉ࣌ฒߦͰؔ࿈͢Δ10℃ͷԘ جରΛຒΊΔʂ → Φʔόʔϥοϓ͕͋ΔྖҬͰಉ࣌ʹ࠷దԽʢྫʣ
• ͨ͠ͷ͔ΒMFEߏΛ νΣοΫ͢Δʢ30℃→10℃ʣ ʢHeurisVcʣ • Ԙجର҆ఆతͳGC͔ΒೖΕͯ ΈΔ • ยํͰ·ͳ͍ͷٯʹෆ ҆ఆͳͷ͔Β
• ϧʔϓྖҬAU͔ΒೖΕͯΈΔ • ͨͩ͠ϥϯμϜมͰॱংʹ ༳Β͗ ԹґଘͰతߏ͕̎ͭ͋Δ߹
݁Ռɹthermo-IRESͷσβΠϯ 42℃ʢ׆ੑߴ͍ʣ 30℃ʢ׆ੑ͍ʣ Experimentally validated! • RNAifold2TͰީิΛڍ͛Δ • ҎԼͷબج४ͰD1,D2ΛηϨΫτ •
(a)(b)ͷߏΛͱΔʢP > 0.2ʣ • ҙਤ͠ͳ͍ߏͷ֬ͷ̎ഒҎ্ • ۙͷυϝΠϯ4ͷߏΛෆ҆ఆԽͤ͞ ͳ͍ Activity 42℃/30℃ WT D1 D2 NC Start codon Py tract • FMDV IRES domain 5 • IRESͷCapඇґଘͳ༁42℃ͷߏ ͷํ͕Α͘ಇ͘
IRES׆ੑͷ࣮ݧతݕূํ๏ • Luc/CAT • 35S labelled Luciferase polypepVde • chloramphenicol
acetyl transferase polypepVde • SDS-PAGE → DensitometerͰൃݱྔͷൺΛऔΔ CAT 5’UTR Luciferase Cap
݁Ռɹ༧ଌ݁Ռൺֱ λ phage thermoregulator RF01804 Relative frequency of the cost
function of SD→͋͘Δඞཁͳ͍ʁ Black : RNAifoldͷશղ୳ࡧ Λ࣌ؒߦͬͨͷ
Cost function Flamm, et al. RNA. 2001. G: partition function
→-RTlnp(Ω1)-RTlnp(Ω2) →ͨͩ݁͠Ռ1Ͱ͍ͬͯͨ બج४ʹྨࣅ
(N<130nt) 30min Supplementary શମతͳྻڍRNAiFold2T ղ͚ͨ૯͋·ΓมΘΒͣ ݁Ռɹthermoregulatorͷྻڍ
(N>130nt) 60min Supplementary ݁Ռɹthermoregulatorͷྻڍ શମతͳྻڍRNAiFold2T ղ͚ͨ૯SD͕एׯ্ճΔ
• 24hrΒͤͯɺग़ͤΔ͚ͩީิΛग़͢ • ΦʔμʔͰܻ̎Ҏ্ҧ͏→ྻڍʹڧ͍ʢྻࣅͯΔՄೳੑ͕ߴ͍ʣ 24hr ݁Ռɹthermoregulatorͷྻڍ
• SHAPEʢ࣮ݧతߏղੳʣͷ σʔλΛ͢ͱߏ༧ଌେ ͖͘มԽ͢Δ • কདྷతʹऔΓೖΕ͍ͨ
ίϝϯτ • ΞϧΰϦζϜͱͯ͠طଘͷͷʹ৽ͨͳ੍ΛՃͨ͠ͷ • χον͚ͩͲݫີղ͑Β͍→طଘख๏ͷίετ࣮ؔΠέͯͳ͍ͷͰʁ • ࣮༻తʹ༷ʑͳheurisVc͕ඞཁ • MFEʹ͔͠ʢ༗ޮʹʣ͑ͳ͍ɺߏܭࢉͷ࠶ར༻0 •
͍ͷʹରͯͨ͘͠͞ΜͷީิΛग़ͤΔͱ͜Ζʹ༏Ґੑ • ͋ͱؤு࣮ͬͯݧͱ͍͏ํੑ • ੜֶతʹ໘ന͍τϐοΫʹ͑Δʢͨʣ͕ධՁ͞Εͨͱ͍͏ҹ