Complex cellular logic computation using ribocomputing devices. Output signals. Output protein. Output gene Gene OFF. NOT logic.

Transcription

1 LETTER doi:1.138/nture23271 Complex ellulr logi omputtion using rioomputing devies lexnder. Green 1,2 *, Jongmin Kim 1,3 *, Duo M 2, Pmel. Silver 1,3, Jmes J. Collins 1,4,5 & Peng Yin 1,3 Syntheti iology ims to develop engineering-driven pprohes to the progrmming of ellulr funtions tht ould yield trnsformtive tehnologies 1. Syntheti gene iruits tht omine DN, protein, nd RN omponents hve demonstrted rnge of funtions suh s istility 2, osilltion 3,4, feedk 5,6, nd logi pilities However, it remins hllenging to sle up these iruits owing to the limited numer of designle, orthogonl, high-performne prts, the empiril nd often tedious omposition rules, nd the requirements for sustntil resoures for enoding nd opertion. Here, we report strtegy for onstruting RN-only nnodevies to evlute omplex logi in living ells. Our rioomputing systems re omposed of de-novo-designed prts nd operte through preditle nd designle se-piring rules, llowing the effetive in silio design of omputing devies with presried onfigurtions nd funtions in omplex ellulr environments. These devies operte t the post-trnsriptionl level nd use n extended RN trnsript to o-lolize ll iruit sensing, omputtion, signl trnsdution, nd output elements in the sme self-ssemled moleulr omplex, whih redues diffusion-medited signl losses, lowers metoli ost, nd improves iruit reliility. We demonstrte tht rioomputing devies in Esherihi oli n evlute twoinput logi with dynmi rnge up to 9-fold nd sle them to four-input ND, six-input OR, nd omplex 12-input expression (1 ND 2 ND NOT 1*) OR (B1 ND B2 ND NOT B2*) OR (C1 ND C2) OR (D1 ND D2) OR (E1 ND E2). Suessful opertion of rioomputing devies sed on progrmmle RN intertions suggests tht systems employing the sme design priniples ould e implemented in other host orgnisms or in extrellulr settings. wide vriety of syntheti iologil iruits hve een onstruted to endow ells with funtions nlogous to those of eletroni iruits. long-term gol of these efforts hs een to develop iologil iruit design strtegies tht will enle ellulr funtion to e progrmmed with the sme ese with whih we progrm eletroni omputers. This oneptul frmework hs motivted efforts to develop lirries of well-hrterized, modulr, nd omposle iologil prts tht, in priniple, n e ssemled to onstrut new types of iruitry in living ells. Furthermore, it hs spurred the doption of lyered iruit designs 11,16,17 in whih the outputs of si two-input iruit elements re fed forwrds into other logi elements in the next lyer. lthough sustntil dvnes hve een mde y using insultion strtegies nd dvned omputer progrms 15 to redue sensitivity to ontext, hllenges remin to further sle up syntheti iologil iruits. Tking inspirtion from the sophistited iruits developed for DN omputing nd self-ssemly in test tues nd dvnes in RN syntheti iology 17,24,25, we hve developed RN-only iruits in teri tht enle omplex intrellulr omputtions to e rried out in single iruit lyer. These iruits hve vrious dvntges for sling up. First, these rioomputing devies utilize progrmmle RN moleules with de-novo-designed prts nd presried intertion rules, llowing effetive in silio designs. Seond, they re omposed of networks of preisely designed syntheti RNs nd funtion purely t the post-trnsriptionl level with no intermedite trnsriptionl Input signls Input RN network OR logi Signl proessing Gte RN: o-lolized sensing nd output Sensor modules ND logi Output gene NOT logi Output signls Output protein Toehold swith: RN sensor Swith RN Trigger RN X X* X* X Toehold swith for ND logi Swith RN Trigger RN Y Y* Y* Y Y* Y Riosome Riosome ON Detivtion Y* Y ON Figure 1 In vivo omputtion using syntheti rioomputing devies., Rioomputing devies use RN moleules s input signls nd protein s the output signl. Signl proessing is rried out y gte RN tht o-lolizes sensing nd output modules. ND, OR, nd NOT logi results from self-ssemly of input nd gte RNs in the devie., Shemti of the toehold swithes tht form the RN sensing elements of the gte RN. The riosoml inding site () nd strt odon () of the swith RN re exposed upon trigger RN inding to tivte trnsltion. X nd X* re omplementry sequenes., Shemti of toehold swithes optimized for ND logi. These toehold swithes retin wek hirpin upon tivtion y the trigger RN tht still llows effiient trnsltion y riosome. 1 Wyss Institute for Biologilly Inspired Engineering, Hrvrd University, Boston, Msshusetts 2115, US. 2 Biodesign Center for Moleulr Design nd Biomimetis, Biodesign Institute nd Shool of Moleulr Sienes, rizon Stte University, Tempe, rizon 85287, US. 3 Deprtment of Systems Biology, Hrvrd Medil Shool, Boston, Msshusetts 2115, US. 4 Institute for Medil Engineering nd Siene, Deprtment of Biologil Engineering, nd Syntheti Biology Center, Msshusetts Institute of Tehnology, Cmridge, Msshusetts 2139, US. 5 Brod Institute of MIT nd Hrvrd, Cmridge, Msshusetts 2142, US. *These uthors ontriuted eqully to this work. 3 UST 217 VOL 548 NTURE Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

2 RESERCH LETTER or trnsltionl steps, minimizing delys nd improving the reliility of signl trnsdution. Third, these devies tke dvntge of o- loliztion to integrte multiple iruit funtions within single trnsript termed gte RN. Implementtion of o-lolized iruit elements enhnes signl propgtion to the output gene nd sustntilly dereses the geneti footprint of the rioomputing devie y enling one gte RN to omplish tsks tht would otherwise require multiple independent RNs. The generl rhiteture of the rioomputing devies is illustrted shemtilly in Fig. 1. network of progrmmed RNs provides input signls to the iruit nd the output signl is protein tht is trnslted upon tivtion of the gte RN. The gte RN omprises the entrl signl-proessing element of the devie, employing modulr sensor domins to detet the self-ssemly stte of the input RN network. intert with themselves nd the gte RN through preditle se-piring. Inputs tht ind to the individul sensor domins on the gte RN n independently trigger protein prodution nd thus re used for OR logi opertions. n intert with one nother oopertively to tivte the gte RN for ND logi or they n inhiit one nother for NOT logi. The sensing modules within the gte RN re tken from reently developed syntheti trnsltionl regultors lled toehold swithes 17. Toehold swithes trnslte n output gene only if ognte trigger RN is expressed in the ell (Fig. 1). The trigger RN inds to swith RN with trnsltion-repressing hirpin struture vi single-strnded toehold region. Trigger inding uses the swith RN stem to unwind, whih exposes the riosoml inding site () nd the strt odon to tivte trnsltion of the output gene. Toehold swith designs optimized for evluting ND logi were lso developed for rioomputing devies (Fig. 1, Extended Dt Fig. 1 nd Supplementry Informtion). These devies feture design in whih the trigger RN unwinds only the lower portion of the swith RN stem to redue trnsltionl lekge. lthough the remins enlosed within stem-loop fter trigger inding, the stem-loop is engineered to e suffiiently wek to llow riosome inding nd strong trnsltion (see Supplementry Informtion nd Extended Dt Fig. 1, ). We initilly onstruted rioomputing iruits tht ould evlute two-input OR, two-input ND, nd ND (NOT B) opertions, whih onstitute funtionlly omplete set of Boolen logi opertors. Gte RNs for two-input OR logi employed two toehold swith sensor modules ontented upstrem of the sequene of output (Fig. 2, Extended Dt Fig. 2). The swith modules nd sequenes were pled in the sme reding frme nd seprted y short single-strnded regions designed to not enode in-frme stop odons. Both swith elements within the gte RN n reognize their ognte trigger RN nd unwind its stem to enle reognition y the e i 2-Input OR gte Input Gte RN * Input B B B* 2-Input ND gte Input 1 Input 2 Gte RN u* * 1 * * B* Riosome B Riosome 2 u 2 1 1* 2* ND (NOT B) gte Input : trigger RN u v Input B: detivting RN u* * v* Input + tive gte RN * u Toeholds v Riosome ON 2 2* u u* 1 1* u u* Riosome B* ON ON ON Detivted inputs u* * v* u v Detivted gte RN * (T) B B (IPTG) Figure 2 Two-input rioomputing logi iruits., Two-input OR gte RN omposed of two swith RN hirpins. Eh swith module hs n input RN reognition site nd its own nd strt odon. Input RN inding unwinds the orresponding swith stem to tivte trnsltion., Flow ytometry mesurements of the two-input OR gte iruit for ognte nd non-ognte inputs., d, outputs for the two-input OR gte on liner () nd logrithmi (d) sles. e, two-input ND gte onstruted from two input RNs tht ind to yield omplete trigger RN. f, Flow ytometry mesurements of the two-input ND iruit under four omintions of input RNs. g, h, The truth tle for the ND omputtion on liner (g) nd logrithmi (h) sles. i, Operting f j 1 2 Normlized ell ounts Normlized ell ounts Normlized ell ounts + B B + X + Y X + Y () fluoresene (.u.) Y 2 + X X + Y (Non-ognte inputs) fluoresene (.u.) T + IPTG IPTG T No induers fluoresene (.u.) 1, g k ,2 1, B T 1 1 IPTG 1 1 d 13 h l B T 1 1 IPTG 1 1 mehnism of the ND (NOT B) iruit in whih detivting RN (input B) uses diret hyridiztion or strnd displement to olish trigger RN (input ) tivity. j, Flow ytometry histogrms of the ND (NOT B) iruit with hemil induers T nd IPTG. k, l, ON/ levels for the ND (NOT B) iruit on liner (k) nd logrithmi (l) sles. ws determined from the geometri men fluoresene of ells mesured vi flow ytometry 4 h fter indution of RN expression. Reltive errors for were otined y dding the reltive errors of ON nd fluoresene in qudrture. Errors for ON nd sttes re the s.d. of three iologil replites. sttes were tken from the null-input se with no ognte RN expressed. 118 NTURE VOL UST Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

3 LETTER RESERCH 4-input ND gte Normlized ell ounts [1,2,3,4] [1,1,1,1] [1,1,1,] [1,1,,1] [1,1,,] [1,,1,1] [1,,1,] [1,,,1] [1,,,] [,1,1,1] [,1,1,] [,1,,1] [,1,,] [,,1,1] [,,1,] [,,,1] [,,,] fluoresene (.u.) d 6-input OR gte e f B C D E F * B* C* D* E* F* Normlized ell ounts Input F Input E Input D Input C Input B Input Deoy Z Deoy Y Deoy X Deoy W Deoy V Deoy U fluoresene (.u.) Figure 3 Multi-input rioomputing ND nd OR iruits., Shemti of the input RN intertion used in four-input ND gte., Flow ytometry mesurements of the four-input ND gte., ON/ for the ND gte truth tle showing ninefold signl inrese upon expression of ll four required inputs. Inset, on logrithmi sle. d, Shemti of the six-input OR gte RN with six sensor modules. e, Flow ytometry mesurements of the six-input OR B C D E F U V W X Y Z BCDEFUVW XYZ Input RNs Deoy RNs gte. f, for the OR gte showing tht ognte inputs provide t lest 12-fold inrese in expression. Inset, on logrithmi sle. ws determined from the geometri men fluoresene of ells mesured vi flow ytometry 4 h fter indution of RN expression. Reltive errors for were otined y dding the reltive errors of the ON nd fluoresene in qudrture. Errors for ON nd sttes re the s.d. of three iologil replites. riosome. One the riosome inds to the nd egins trnsltion, it n sn through the gte trnsript, unwind ny downstrem swith hirpins, nd ontinue with trnsltion of. Thus, ny ognte RN n tivte trnsltion from the gte RN to perform OR logi. Rioomputing iruits were first evluted in E. oli BL21 Str DE3, n RNse-defiient strin, with T7 RN polymerse expression indued y isopropyl β -d-1-thiogltopyrnoside (IPTG; see Methods nd Supplementry Informtion for full experimentl detils). Unless otherwise noted, nlogous onditions were employed for testing the other iruits herein. The two-input gte RN ws o-expressed with different omintions of input RNs, nd B, nd deoy RNs, X nd Y (designed for other rioomputing devies), to determine expression in flow ytometry (Fig. 2). Cognte inputs nd B produed roust expression with inresed signl output in the presene of oth inputs, wheres the deoys yielded very low fluoresene output, resulting in ON/ levels over 4-fold (Fig. 2, d). To implement rioomputing ND logi, we divided the trigger RN sequene of toehold swith evenly into two seprte input RNs (Fig. 2e) nd used toehold swithes optimized to ompute ND expressions. When either input RN is expressed, it is inple of tivting the swith euse neither trigger su-sequene lone n unwind the repressing hirpin. Complementry inding domins (u nd u* in Fig. 2e) were designed etween the two input RN speies to enle them to hyridize nd form omplete trigger sequene when expressed (see Extended Dt Fig. 2 for design shemti, Extended Dt Fig. 3, for dimensioning study). nlogous ssoitive trigger systems exploiting oopertive self-ssemly hve previously een implemented in vitro using DN 23,26,27. Mesurements of the ND iruit demonstrted very low output in ll three logil FLSE onditions nd 9-fold inrese in expression for the logil TRUE ondition ompred to the null-input se with two non- ognte RNs (Fig. 2f h). Devies tested with non-rnse-defiient E. oli provided levels of 175-fold or more (Extended Dt Fig. 3 f). NOT logil ehviour ws omplished through diret hyridiztion of detivting RN to trigger RN to silene its effet on the gte RN (Fig. 2i, Extended Dt Fig. 2d). The detivting RN n ind diretly to free trigger RNs nd use the extended single-strnded domins of the trigger RN (u nd v in Fig. 2i) s toeholds to disple the trigger fter it hs ound to the gte RN. These repressing systems evlute ND (NOT B) logi nd were tested in E. oli MG1655Pro using the hemil induers nhydrotetryline (T) nd IPTG to express inputs nd B, respetively (see Supplementry Informtion for experimentl detils). fluoresene histogrms (Fig. 2j) showed ler inreses in fluoresene in the logil TRUE se with only the trigger RN expressed nd 19-fold derese with oth inputs expressed (Fig. 2k, l). We next investigted sling of the rioomputing devies y testing iruits with inresing numers of ND nd OR inputs. Four different three-input ND iruits produed orret truth tles, with the est providing t lest 25-fold inrese in for the TRUE stte ompred to ll logil FLSE sttes (Extended Dt Fig. 4 g). For four-input ND gtes, we oserved lower ON stte output s we hllenged in vivo RN self-ssemly with iruit omprising five interting RNs (Fig. 3, Extended Dt Fig. 4i). histogrms from the 16-element truth tle showed ninefold inrese in for the TRUE stte over the null-input se nd t lest sixfold inrese over the most leky FLSE stte (Fig. 3, ). These performne levels re etter thn previous toehold-swith-sed lyered four-input ND gtes 17. Furthermore, they re omprle to previous fourinput ND gte onstruted from lyered trnsription ftors 11. The rioomputing four-input ND system is lso genetilly ompt, requiring only five progrmmed RNs with totl length of 392 nuleotides (nt). Mesurements of seond four-input ND gte nd five-input ND gte re shown in Extended Dt Fig. 4j n. We tested OR gte RNs with inresing numers of inputs (see Extended Dt Fig. 5 for systemti study nd Extended Dt Fig. 6 for four- nd five-input OR gtes). The most omplex gte RN we tested onsisted of six sensor modules nd hd sensor region length of 444 nt (Fig. 3d, Extended Dt Fig. 2). Mesurements of this six-input OR gte reveled low lekge levels generted from six deoy speies nd inreses in expression of t lest 12-fold for the ognte inputs 3 UST 217 VOL 548 NTURE Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

4 RESERCH LETTER 1* (1 ND 2 ND NOT 1*) OR (B1 ND B2 ND NOT B2*) OR (C1 ND C2) OR (D1 ND D2) OR (E1 ND E2) B1 B2* B2 C1 C2 D1 D2 E1 E2 * B* C* D* E* Normlized ell ounts fluoresene (.u.) [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [1,1,1] [1,1,] [1,,1] [1,,] [,1,1] [,1,] [,,1] [,,] [1,1,1] [1,1,] [1,,1] [1,,] [,1,1] [,1,] [,,1] [,,] [E1,E2] [D1,D2] [C1,C2] [B1,B2,B2*] [1,2,1*] n* B C D E inputs inputs n* inputs ND 2 ND NOT 1* B1 ND B2 ND NOT B2* Figure 4 Twelve-input DNF rioomputing iruit., Shemti of the 12-input DNF expression evluted in E. oli., Flow ytometry mesurements show low output for 23 logil FLSE sttes nd t lest tenfold inreses in for the five logil TRUE sttes., ON/ from the DNF iruit under 28 different input RN omintions. Inset, on logrithmi sle. ws determined C1 ND C2 D1 ND D2 E1 ND E2 from the geometri men fluoresene of ells mesured vi flow ytometry 6 h fter indution of RN expression. Reltive errors for were otined y dding the reltive errors of the ON nd fluoresene in qudrture. Errors for ON nd sttes re the s.d. of three iologil replites. sttes were tken from the null-input se of the 1 ND 2 ND NOT 1* luse. (Fig. 3e, f). Despite the strong overll signl for logil TRUE onditions, we oserved sustntil vritions in depending on the input RN expressed. These vritions ould e ttriuted to the effets of downstrem gte RN seondry struture on riosome proession nd the dditionl mino ids inorported into the output protein for the more upstrem sensor modules. We lso evluted gte RNs regulting other output proteins (Extended Dt Fig. 7 d) nd implemented n 11-input iruit in whih two gte RNs were expressed simultneously (Extended Dt Fig. 7e g). OR gte iruits were lso tested in non-rnse-defiient E. oli strins nd using different promoters (Extended Dt Fig. 8). Lstly, we onstruted iruits tht omined ND, OR, nd NOT shemes to ompute expressions in disjuntive norml form (DNF). DNF expressions n e used to evlute ny Boolen logi expression nd onsist of ND nd NOT opertions tht provide inputs for OR opertions. The most omplex expression we evluted ws the 12-input RN omputtion (1 ND 2 ND NOT 1* ) OR (B1 ND B2 ND NOT B2* ) OR (C1 ND C2) OR (D1 ND D2) OR (E1 ND E2) (Fig. 4). Inputs 1* nd B2* re omplementry to 1 nd B2, respetively. We found tht this iruit funtioned roustly in vivo, displying ler signl differenes etween TRUE nd FLSE sttes for 28 input onditions tested (Fig. 4). fter 6 hours of IPTG indution, for logil TRUE onditions rnged from 22-fold to 41-fold higher thn the null-input se, with low signl lekge for multiple omintions of non-ognte RNs (Fig. 4). This 12-input single-lyer rioomputing iruit evlutes logi expression tht would require eleven two-input or signl inversion opertions in onventionl lyered iruit implementtion. Mesurements of eightnd ten-input DNF rioomputing iruits re shown in Extended Dt Figs 9, 1. We hve developed strtegy for onstruting RN-sed iologil iruits tht exploits the progrmmle se-piring properties of RN nd uses o-lolized sensing nd output modules to enle omplex trnsltionl regultion (see Supplementry Informtion for extended disussion). These rioomputing devies re enoded in smll geneti footprint ompred to typil protein-sed iruits nd hve the potentil to e sled up using the lrge sequene spe fforded y RN. The rioomputing devie rhiteture requires self-ssemly etween the input RNs for ND nd NOT logi, nd hene imposes some sequene dependenies on these RNs. The use of o- lolized gte RN requires dditionl N-terminl residues in the output protein, whih ould interfere with its funtion. Inorportion of rioomputing devies into sophistited lyered iruits, suh s those mde possile with dvned geneti iruit design tools 15, will require systems tht n provide RNs s output speies. This funtionlity n e implemented using gte RNs to regulte RN polymerses or trnsription ftors, s hs een demonstrted previously for toehold swithes 17,28. Integrtion of mrn-sensing rioomputing iruits with 12 NTURE VOL UST Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

5 LETTER RESERCH pper-sed syntheti iology systems ould improve the roustness nd reliility of these dignosti tools when they re deployed in the field 28,29. Detetion of mrns nd other nturlly ourring RNs s inputs for ND logi, however, will require dditionl syntheti RNs to interfe ntive trnsripts. Finlly, the effetive use of preditle nd roust se-piring intertions in rioomputing devies suggests tht this strtegy ould e pplied in prokryoti hosts eyond E. oli. Online Content Methods, long with ny dditionl Extended Dt disply items nd Soure Dt, re ville in the online version of the pper; referenes unique to these setions pper only in the online pper. Reeived 3 July 216; epted 8 June 217. Pulished online 26 July Cmeron, D. E., Bshor, C. J. & Collins, J. J. rief history of syntheti iology. Nt. Rev. Miroiol. 12, (214). 2. Grdner, T. S., Cntor, C. R. & Collins, J. J. Constrution of geneti toggle swith in Esherihi oli. Nture 43, (2). 3. Elowitz, M. B. & Leiler, S. syntheti osilltory network of trnsriptionl regultors. Nture 43, (2). 4. Dnino, T., Mondrgón-Plomino, O., Tsimring, L. & Hsty, J. synhronized quorum of geneti loks. Nture 463, (21). 5. Bshor, C. J., Helmn, N. C., Yn, S. & Lim, W.. Using engineered sffold intertions to reshpe MP kinse pthwy signling dynmis. Siene 319, (28). 6. Dniel, R., Ruens, J. R., Srpeshkr, R. & Lu, T. K. Syntheti nlog omputtion in living ells. Nture 497, (213). 7. Rinudo, K. et l. universl RNi-sed logi evlutor tht opertes in mmmlin ells. Nt. Biotehnol. 25, (27). 8. Win, M. N. & Smolke, C. D. Higher-order ellulr informtion proessing with syntheti RN devies. Siene 322, (28). 9. Tmsir,., Tor, J. J. & Voigt, C.. Roust multiellulr omputing using genetilly enoded NOR gtes nd hemil wires. Nture 469, (211). 1. Xie, Z., Wrolewsk, L., Prohzk, L., Weiss, R. & Benenson, Y. Multi-input RNi-sed logi iruit for identifition of speifi ner ells. Siene 333, (211). 11. Moon, T. S., Lou, C., Tmsir,., Stnton, B. C. & Voigt, C.. ti progrms onstruted from lyered logi gtes in single ells. Nture 491, (212). 12. usländer, S., usländer, D., Müller, M., Wielnd, M. & Fussenegger, M. Progrmmle single-ell mmmlin ioomputers. Nture 487, (212). 13. Bonnet, J., Yin, P., Ortiz, M. E., Susoontorn, P. & Endy, D. mplifying geneti logi gtes. Siene 34, (213). 14. Tor, J. J. et l. syntheti geneti edge detetion progrm. Cell 137, (29). 15. Nielsen,.. K. et l. ti iruit design utomtion. Siene 352, 7341 (216). 16. Kini, S. et l. CRISPR trnsriptionl repression devies nd lyered iruits in mmmlin ells. Nt. Methods 11, (214). 17. Green,.., Silver, P.., Collins, J. J. & Yin, P. Toehold swithes: de-novodesigned regultors of gene expression. Cell 159, (214). 18. Brih, R. S., Chelypov, N., Johnson, C., Rothemund, P. W. K. & dlemn, L. Solution of 2-vrile 3-ST prolem on DN omputer. Siene 296, (22). 19. Chen, Y.-J., Groves, B., Must, R.. & Seelig, G. DN nnotehnology from the test tue to the ell. Nt. Nnotehnol. 1, (215). 2. Qin, L., Winfree, E. & Bruk, J. Neurl network omputtion with DN strnd displement sdes. Nture 475, (211). 21. Elz, J. et l. DN omputing iruits using lirries of DNzyme suunits. Nt. Nnotehnol. 5, (21). 22. Lke,., Shng, S. & Kolpshhikov, D. M. Moleulr logi gtes onneted through DN four-wy juntions. ngew. Chem. Int. Ed. 49, (21). 23. Zhu, J., Zhng, L., Dong, S. & Wng, E. Four-wy juntion-driven DN strnd displement nd its pplition in uilding mjority logi iruit. CS Nno 7, (213). 24. Rodrigo, G., Lndrin, T. E. & Jrmillo,. De novo utomted design of smll RN iruits for engineering syntheti rioregultion in living ells. Pro. Ntl d. Si. US 19, (212). 25. Chppell, J., Tkhshi, M. K. & Luks, J. B. Creting smll trnsription tivting RNs. Nt. Chem. Biol. 11, (215). 26. Chen, X. Expnding the rule set of DN iruitry with ssoitive toehold tivtion. J. m. Chem. So. 134, (212). 27. Genot,. J., Bth, J. & Turerfield,. J. Comintoril displement of DN strnds: pplition to mtrix multiplition nd weighted sums. ngew. Chem. 52, (213). 28. Prdee, K. et l. Pper-sed syntheti gene networks. Cell 159, (214). 29. Prdee, K. et l. Rpid, low-ost detetion of Zik virus using progrmmle iomoleulr omponents. Cell 165, (216). Supplementry Informtion is ville in the online version of the pper. knowledgements This work ws supported y NIH Diretor s New Innovtor nd Trnsformtive Reserh wrds (1DP2OD7292, 1R1EB18659), n ONR Young Investigtor Progrm wrd (N ) nd grnts (N , N , N , N ), NSF CREER nd Expedition in Computing wrds (CCF154898, CCF ) nd grnts (CCF , ERSynBio ), nd Wyss Institute Moleulr Rootis Inititive support to P.Y.; DRP Living Foundries grnt (HR1112C61) to P..S., P.Y., nd J.J.C.; n ONR MURI Progrm grnt, DTR grnt (HDTR ), nd Pul G. llen Frontiers Group funds to J.J.C.; nd n rizon Biomedil Reserh Commission New Investigtor wrd, n lfred P. Slon Reserh Fellowship (FG ), nd rizon Stte University funds to..g. J.K. knowledges Wyss Institute Diretor s Cross-Pltform Fellowship. uthor Contriutions..G. oneived the study, designed nd performed the experiments, nlysed the dt, supervised D.M. nd wrote the pper. J.K. oneived the study, designed nd performed the experiments, nlysed the dt nd wrote the pper. D.M. performed experiments nd nlysed the dt. P..S. supervised the study. J.J.C. supervised the study. P.Y. oneived nd supervised the study, interpreted the dt, nd wrote the pper. ll uthors reviewed nd pproved the mnusript. uthor Informtion Reprints nd permissions informtion is ville t The uthors delre ompeting finnil interests: detils re ville in the online version of the pper. Reders re welome to omment on the online version of the pper. Pulisher s note: Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Correspondene nd requests for mterils should e ddressed to P.Y. (py@hms.hrvrd.edu) or..g. (lexgreen@su.edu). 3 UST 217 VOL 548 NTURE Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

6 RESERCH LETTER METHODS Strins nd growth onditions. The following E. oli strins were used in this study: BL21 Str DE3 (F ompt hsds B (r B m B ) gl dm rne131 (DE3); Invitrogen), BL21 DE3 (F ompt hsds B (r B m B ) gl dm (DE3); Invitrogen), MG1655Pro (F λ ilvg- rf-5 rph-1 Sp R lr tetr), nd DH5α (end1 re1 gyr96 thi-1 glnv44 rel1 hsdr17(r K m K + ) λ ; Invitrogen). ll strins were grown in LB medium t 37 C with pproprite ntiiotis: mpiillin (5 µ g ml 1 ), spetinomyin (25 µ g ml 1 ), hlormpheniol (17 µ g ml 1 ), nd knmyin (3 µ g ml 1 ). Plsmid onstrution. Plsmids were onstruted using PCR nd Gison ssemly. DN templtes for expressing gte nd input RNs were ssemled from single-strnded DNs purhsed from Integrted DN Tehnologies. The syntheti DN strnds were mplified vi PCR to form doule-strnded DNs. The resulting DNs were then inserted into plsmid kones using 3-p homology domins vi Gison ssemly 3. ll plsmids were loned in the E. oli DH5α strin nd vlidted through DN sequening. Bkones for the plsmids were tken from the ommeril vetors pet15, pcolduet, pcdfduet, nd pcycduet (EMD Millipore). mut3-sv ws used s the reporter for the gte plsmids. This is mut3 with n SV degrdtion tg 31. mcherry nd erulen were lso used s reporter proteins for seleted OR gte plsmids. Sequenes of elements ommonly used in the plsmids re provided in Supplementry Tle 1. Sequenes for ll rioomputing devies, ND-omputing toehold swithes, nd deoy RNs re ontined in Supplementry Tles 2 9. Rioomputing devie indution onditions. Unless otherwise noted, RNs in the ND, OR, nd DNF networks were expressed using T7 RN polymerse in BL21 Str DE3, n RNse-defiient strin, with the T7 RN polymerse indued with the ddition of IPTG. Two-input ND gtes nd the 6-input OR gte were lso evluted in BL21 DE3, non-rnse-defiient strin, with the T7 RN polymerse indued with IPTG. ND (NOT B) nd 6-input OR gte iruits employing the endogenous E. oli RN polymerse were evluted in MG1655Pro using onstitutive promoters or indution vi IPTG nd/or T, s required. For ll strins, ells were grown overnight in 96-well pltes with shking t 9 r.p.m. nd 37 C. Overnight ultures were then diluted 1-fold into fresh medium nd returned to shking (9 r.p.m., 37 C). fter 8 min, BL21 Str DE3 nd BL21 DE3 ultures were indued with.1 mm IPTG, nd MG1655Pro ultures were indued with the pproprite omintion of 1 mm IPTG nd 5 ng ml 1 T. Cells were returned to the shker (9 r.p.m., 37 C) nd mesured t the speified times post-indution. Flow ytometry mesurements nd nlysis. Flow ytometry mesurements were performed using BD LSRFortess ell nlyser with high-throughput smpler. Prior to smpling, ells were diluted y ftor of 65 into phosphte-uffered sline. Cells were deteted using forwrd stter (FSC) trigger nd t lest 1, ells were reorded for eh mesurement. Cell popultions were gted ording to their FSC nd side stter (SSC) distriutions s desried previously 17, nd the fluoresene levels of these gted ells were used to mesure iruit output. fluoresene histogrms yielded unimodl popultion distriutions nd the geometri men ws employed to extrt the verge fluoresene ross the pproximtely log-norml fluoresene distriution from t lest three iologil replites. levels were then evluted y tking the verge fluoresene from given omintion of input RNs nd dividing it y the fluoresene from the null-input se with no ognte input RNs expressed. Cellulr utofluoresene ws not sutrted efore determining the ON/ rtio. The sme fluoresene dt nlysis proedures were used for OR gtes using mcherry nd erulen s reporter proteins. No sttistil methods were used to predetermine smple size. The experiments were not rndomized nd the investigtors were not linded to llotion during experiments nd outome ssessment. Dt vilility. The uthors delre tht the min dt supporting the findings of this study re ville within the pper nd its Supplementry Informtion files. Soure Dt for Figs 2 4 re provided with the pper. ll other dt supporting the findings of this study re ville from the orresponding uthors on request. 3. Gison, D. G. et l. Enzymti ssemly of DN moleules up to severl hundred kiloses. Nt. Methods 6, (29). 31. ndersen, J. B. et l. New unstle vrints of green fluoresent protein for studies of trnsient gene expression in teri. ppl. Environ. Miroiol. 64, (1998). 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

7 LETTER RESERCH ND-Computing Toehold Swith (CTS) Design with Single Trigger Swith RN 14-nt loop Hlf-trigger juntion site Type I Design 6-p upper stem 12-p lower * stem Swith RN Hlf-trigger juntion site 12-nt loop * Type II Design 5-p upper stem 11-p lower stem 16-nt toehold Trigger RN 5 hirpin 21-nt linker 28-nt intertion domin 47-nt termintor 15-nt toehold Trigger RN 5 hirpin 21-nt linker 26-nt intertion domin 47-nt termintor tivted swith Linker tivted swith Linker G -linker su-sequene G -linker su-sequene Proposed CTS tivtion Mehnism Swith RN Trigger RN Low GC stem * * * * Riosome ON Trigger inding disrupts lower stem. is repressed only through low-gc-ontent stem. Upper stem is suffiiently wek (5 to 6 p) to open trnsiently. Riosome inding lso enourges stem opening. fter upper stem unwinds fully, the riosome n initite trnsltion nd produe the output protein. CTS Lirry Dynmi Rnge 1,8 1,6 1,4 1,2 1, Extended Dt Figure 1 Design, tivtion mehnism, nd hrteriztion of ND-omputing toehold swithes (CTS)., Nuleotide-level shemtis of the Type I nd Type II CTS systems (see Supplementry Informtion Setion 1.2 for disussion). Green nd ornge ses speify the output sequene nd the ommon 21-nt linker sequene used, respetively. Blk ses mrk iologilly onserved sequenes, suh s the, strt odon, nd trnsriptionl termintor. White ses represent those tht n dopt ny sequene sujet to seondry struture onditions in NUPCK. Progrmmed hyridiztion domins etween different strnds re speified y olour., The proposed CTS tivtion mehnism in whih the trigger RN Type I Type II prtilly unwinds the swith RN stem. The remining wek stem, with low GC ontent, n intert with the riosome to initite trnsltion., levels mesured for the CTS systems employed in this study. levels were determined from the geometri men fluoresene of ells mesured vi flow ytometry 3 h fter indution with.1 mm IPTG. Reltive errors for the swith ON/ rtios were otined y dding the reltive errors of the swith ON nd fluoresene mesurements in qudrture. Reltive errors for ON nd sttes re from the s.d. of three iologil replites. Flow ytometry dt were produed using the sme proedure nd the sme numer of iologil replites in susequent Extended Dt figures. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

8 RESERCH LETTER Gte RN * 2-Input OR Gte B* * Linker 6-Input OR Gte Using First-rtion Toehold Swithes (444-nts) B* * B* C* D* E* F* * Linker B* Linker C* Linker D* Linker E* Linker F* 2-Input ND Ciruit with CTS 5 hirpin Input 1 u* 1 5 hirpin Input 2 2 u Gte tivted ND Gte 1* 2* d ND (NOT B) Ciruit Input Intertion Input : Trigger RN Input B: Detivting RN u v 5 hirpin 5 hirpin u* * v* Extended Dt Figure 2 Nuleotide-level shemtis of rioomputing devies., Seondry struture of the two-input OR gte used in Fig. 2 d., Seondry struture of the six-input OR gte RN used for iruits in Fig. 3d f nd Extended Dt Figs 7, 8., Shemti of two-input ND gte using Type I CTS system. 1 nd 2 domins re 14-nt hlves of 28-nt-long omplete trigger RN. d, Shemti of the ND (NOT B) iruit design. The ND (NOT B) system design fetures nerly perfetly omplementry trigger (input ) nd detivting (input B) RN strnds used in Fig. 2i l. For ll pnels, lk ses mrk iologilly Detivted inputs nd B onserved sequenes, suh s the nd strt odon. White ses represent those tht n dopt ny sequene sujet to seondry struture onditions in NUPCK. Grey ses re those whose sequenes were originlly determined on the sis of seondry struture onsidertions for the prentl toehold swithes nd were left onstnt during the design of RN iruit elements. The remining progrmmed hyridiztion domins etween different strnds re speified y olour. Input RN shemtis re trunted just efore the trnsriptionl termintor sequene. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

9 LETTER RESERCH 2-Input ND Gte Input Overlp Study Input 1 Input 2 Gte RN 1,2 1, u* 1 2 1* 2* u BL21 Str DE3 BL21 DE3 2 2* u u* 1 1* 2 u u* 1 Riosome d ON BL21 Str DE3 BL21 DE3 u 2 u* u domin length (nts) Melting temperture ( C) e 3 25 BL21 Str DE3 BL21 DE3 f 1 2 BL21 Str DE3 BL21 DE Extended Dt Figure 3 Systemti study of ND gte iruit overlp domin lengths nd omprison of two-input ND rioomputing devies in different strins., n erly two-input ND gte ws onstruted from stndrd toehold swith y dividing the trigger evenly into two 15-nt domins, 1 nd 2. Overlp domins u nd u* were designed to use the two input RNs to hyridize nd form n tive trigger., domin u ws used to vry the region omplementry to u* nd mesure its effet on expression levels. rtios (left xis) vry s funtion of the u domin length. The onset of sustntil expression oinides with the melting temperture of u u* hyridiztion rising ove 37 C (right xis). f, Comprison of two-input ND rioomputing devies in RNse-defiient E. oli BL21 Str DE3 nd non-rnse-defiient E. oli BL21 DE3., d, on liner () nd logrithmi (d) sles mesured for the two-input ND gte from Fig. 2e h. e, f, on liner (e) nd logrithmi (f) sles mesured for seond two-input ND gte with n identil design ut different RN sequenes. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

10 RESERCH LETTER 3-Input ND Gte 3-Input ND Ciruit [1,2,3] d Input Intertion [1,1,1] 5 e h k Input ND Gte Input ND Gte f i m Normlized ell ounts fluoresene (.u.) 4-Input ND Ciruit Input Intertion [1,1,] [1,,1] [1,,] [,1,1] [,1,] [,,1] [,,] j g l 5-Input ND Ciruit Input Intertion n Extended Dt Figure 4 Three-, four-, nd five-input ND gte systems., rl shemti for three-input ND gte with output., Nuleotide-level shemti of the tivted trigger omplex for the three-input ND logi iruits., Flow ytometry mesurements from the three-input ND gte with the truth tle shown in d. d g, Truth tles for four different three-input ND gtes. h, rl shemti for four-input ND gte with output. i, Nuleotide-level shemti of the tivted trigger omplex for the four-input ND logi iruits. j, Truth tle for n dditionl four-input ND gte. k, rl shemti for the five-input ND gte with output. l, Nuleotide-level shemti of the tivted trigger omplex for the five-input ND logi iruit. m, Liner-sle truth tle for the five-input ND gte, showing sttistilly signifint differene etween logil TRUE nd logil FLSE onditions (P <.3, Welh s unequl vrines t-test). n, Logrithmi-sle truth tle for the five-input ND gte. Insets of d g, j show logrithmi-sle plots of for the devies. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

11 LETTER RESERCH 4-input OR gtes with systemti vritions in seondry struture B C D Version 1: Originl CTS stems Linker Linker Linker Version 2: 1-2 fewer se pirs per stem Linker Linker Linker Version 3: 2-3 fewer se pirs per stem d Fluoresene (.U.) 15 x 14 Version 1 Version 2 1 Version 3 5 2, 1,6 1,2 8 4 Version 1 Version 2 Version 3 B C D W X Y Z Linker Linker Linker B C D W X Y Z Fluoresene (.U.) Deresed ON stte (2 vs 1) Inresed stte (2 vs 3) B C D W X Y Z Version 1 Version 2 Version 3 B C D W X Y Z Extended Dt Figure 5 Systemti study of gte RN performne s funtion of seondry struture., Nuleotide-level shemtis of three four-input OR gte versions feturing smll hnges in seondry struture nd sequene. Version 1 dopts the originl seondry strutures of the CTS swith RNs. Version 2 differs from the first gte RN t the six positions mrked in red, whih wekens the hirpin seondry struture. Version 3 hs n dditionl mismth in the hirpin lower stem mrked in lue. ll other ses remin the sme ross the three gte RNs.,, fluoresene levels mesured for the gte RN versions for pnel of eight RN triggers shown in liner () nd logrithmi () sles. d, rtios lulted for the three gte RNs. Gte RN version 2 provides the est omintion of low lekge nd high ON stte expression. Inset, logrithmi-sle plot of iruit ON/ levels. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

12 RESERCH LETTER 4-Input OR Gte Using CTS B C D * B* C* D* Input OR Gte Using CTS 3 B C D E * B* C* D* E* B C D W X Y Z B C D E X Y Z B C D W X Y Z B C D E X Y Z Extended Dt Figure 6 Four- nd five-input OR gte systems., Liner- nd logrithmi-sle plots of ON/ levels of four-input OR gte onstruted from CTS hirpin modules (shemti, left)., Liner- nd logrithmi-sle plot of ON/ levels of five-input OR gte onstruted from CTS devies (shemti, left). Both OR logi gtes were mesured 3 h fter indution of T7 RN polymerse expression. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

13 LETTER RESERCH ON/ mcherry e B C D E X Y Z 11-Input Dul OR Gte Ciruit ON/ mcherry Inputs B C D E X Y Z mcherry Inputs 1 B1 C1 D1 E1 F1 2 B2 C2 D2 E2 ON/ Cerulen B C D E X Y Z d ON/ Cerulen B C D E X Y Z f 1, mcherry ON/ mcherry 2 2 B C mcherry D EF 12 U V WX Y Z g 1 3 mcherry Single-Input Cses Dul-Input Cses 1 2 ON/ B C mcherry D EF 12 U V WX Y Z Single-Input Cses Extended Dt Figure 7 Gte RN regultion of mcherry nd erulen outputs with five-input OR gtes nd n 11-input dul OR gte iruit.,, ON/ mcherry rtio for five-input CTS-sed OR gte on liner () nd logrithmi () sles., d, ON/ erulen rtio for five-input CTS-sed OR gte on liner () nd logrithmi (d) sles. e, six-input OR gte ws used to regulte nd five-input Dul-Input Cses CTS-sed OR gte ws used to regulte mcherry. f, g, ON/ rtios of the gte RNs on liner (f) nd logrithmi (g) sles. Comintions of one- or two-input or deoy RNs were expressed s speified y the filled green ( inputs), red (mcherry inputs), nd lk (deoys) irles elow eh pnel. ll iruit responses were mesured vi flow ytometry 4 h fter IPTG indution. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

14 RESERCH LETTER 1,2 1, BL21 Str DE3 BL21 DE BL21 Str DE3 BL21 DE B C D E F V W X Y Z 1 1 B C D E F V W X Y Z 25 d B C D E F V W X Y Z Extended Dt Figure 8 Comprison of six-input OR gte rioomputing devies mesured in RNse-defiient E. oli (BL21 Str DE3) nd non-rnse-defiient E. oli (BL21 DE3, MG1655Pro).,, rtios mesured for the devie using T7 RN polymerse in BL21 Str DE3 nd BL21 DE3 ells on liner () nd logrithmi () sles. Gte nd input RNs were expressed using the 1-1 B C D E F V W X Y Z T7 RN polymerse nd mesured 4 h fter indution with IPTG., d, rtios otined from the OR gte using E. oli RN polymerse in MG1655Pro ells on liner () nd logrithmi (d) sles. Gte nd input RNs were expressed using the E. oli RN polymerse nd mesured 4 h fter indution of the gte RN with IPTG. Input nd deoy RNs were expressed using onstitutive PN25 promoter. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

15 LETTER RESERCH 8-Input DNF Gte: (1 ND 2) OR (B1 ND B2) OR (C1 ND C2) OR (D1 ND D2) 1 2 B1 B2 C1 C2 D1 D * B* C* D* Normlized ell ounts fluoresene (.u.) [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [D1,D2] [C1,C2] [B1,B2] [1,2] inputs inputs d ND 2 B1 ND B2 C1 ND C2 D1 ND D inputs inputs ND B1 ND B C1 ND C2 Extended Dt Figure 9 Evlution of n eight-input DNF iruit., The eight-input DNF iruit fetures four two-input NDs oupled to the four-input OR gte RN tested in Extended Dt Fig. 6., fluoresene histogrms otined from flow ytometry mesurements of D1 ND D2 the iruit under 16 different omintions of input RNs., d, ON/ levels otined from flow ytometry on liner () nd logrithmi (d) sles. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.

16 RESERCH LETTER 1-Input DNF Gte: (1 ND 2) OR (B1 ND B2) OR (C1 ND C2) OR (D1 ND D2) OR (E1 ND E2) 1 2 B1 B2 C1 C2 D1 D2 E1 E2 6 5 * B* C* D* E* Normlized ell ounts fluoresene (.u.) [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [1,] [,1] [,] [E1,E2] [D1,D2] [C1,C2] [B1,B2] [1,2] inputs inputs d 1 ND 2 B1 ND B2 C1 ND C2 D1 ND D2 E1 ND E inputs inputs ND B1 ND B C1 ND C D1 ND D E1 ND E2 Extended Dt Figure 1 Evlution of 1-input DNF iruit., The 1-input DNF iruit fetures five two-input NDs oupled to the five-input OR gte RN tested in Extended Dt Fig. 6., fluoresene histogrms otined from flow ytometry mesurements of the iruit under 2 different omintions of input RNs., d, levels otined from flow ytometry on liner () nd logrithmi (d) sles. 217 Mmilln Pulishers Limited, prt of Springer Nture. ll rights reserved.