rnai enables biologists 
 to knock down expression of a gene of choice 
 in eukaryotes , providing a powerful tool for probing gene function 
 within endogenous biological circuits . rnai can be 
 activated by exogenous double - stranded rnas that are cleaved by the 
 enzyme dicer to produce sirnas . one strand of the sirna duplex ( the 
 guide strand ) is loaded into the rna - induced silencing complex ( risc ) , 
 where it serves as a recognition domain for recruitment of target 
 mrnas containing the complementary sequence . risc cleaves and releases 
 the mrna for subsequent degradation , enabling a single guide strand 
 to mediate destruction of multiple copies of the mrna silencing target . the conceptual power of rnai follows from its programmability : by 
 changing the sequence of the sirna , it is possible to change the identity 
 of the gene that is targeted for knockdown . using an sirna programmed 
 to silence gene y , conventional rnai 
 implements the unconditional molecular logic ( inset of figure 1 ) : silence gene y. to exert control over the strength 
 and/or timing of gene knockdown , numerous methods have been developed 
 to implement drug - inducible rnai , where the activation ( or inhibition ) 
 of knockdown is made dependent on the presence of a small molecule , 
 using either pretranscriptional protein machinery or 
 post - transcriptional rna machinery . to achieve spatiotemporal control over gene knockdown , we seek to 
 engineer scrnas that mediate conditional rnai corresponding to the 
 conditional molecular logic ( figure 1 ) : if 
 gene x is transcribed , silence independent gene y. this logic is programmable 
 at two levels , with input sequence x controlling the scope of silencing 
 and output sequence y controlling the target of silencing . molecular logic 
 of conditional and conventional rnai . conditional 
 rnai ( if gene x is transcribed , silence independent gene y ) provides 
 a conceptual framework for exerting spatiotemporal control over gene 
 knockdown . toward this end , small conditional rnas ( scrnas ) detection 
 target x and production of a dicer substrate targeting independent 
 mrna  silencing target y. inset : conventional rnai 
 ( silence gene y ) employs constitutively active dicer substrates , making 
 it difficult to control the locus and time of gene knockdown . we consider 
 conditional formation of dicer substrates that are either dsirnas 
 or shrnas . to implement the logic of conditional 
 rnai , our approach is to 
 engineer scrna signal transduction cascades in which hybridization 
 of an scrna to an mrna detection target x initiates 
 downstream conformational changes of one or more scrnas leading to 
 formation of a dicer substrate targeting independent mrna  silencing 
 target y. dicer processing of this substrate then yields an 
 sirna targeting mrna y for destruction . two types of signal transduction 
 must be performed simultaneously to achieve this goal : conditional 
 shape change is required to produce a molecular geometry that is recognized 
 and processed by dicer , and conditional sequence change is required 
 to shift from input sequence x to output sequence y. previous 
 studies have shown that suitable dicer substrates include 
 short hairpin rnas ( shrnas ; 1929-bp stem with a 2-nt 3-overhang ) and so - called dicer - substrate 
 rnas ( dsirnas ; 25-bp duplex with a 2-nt 3-overhang 
 at one end ) . dicer functions as a molecular 
 ruler , measuring from the 2-nt 3-overhang to cleave 2123-nt 
 sirna strands that form a duplex with 2-nt 3-overhangs at 
 both ends . for this reason , we focus on 
 engineering scrna transducers that conditionally assemble shrna or 
 dsirna dicer substrates with a 2-nt 3-overhang at one end 
 of a minimum 19-bp duplex . to mediate conditional rnai via dicer 
 substrate formation , several 
 design requirements for scrna function can be identified a priori : 
 first , the sequence of the detection target x must place no restriction 
 on the sequence of the independent silencing target y. second , in 
 the absence of detection target x , the scrnas should not interact 
 to form the dicer substrate targeting y. third , the scrnas must be 
 capable of detecting a subsequence of a full - length endogenous mrna 
 detection target x. fourth , in response to detection of x , the scrnas 
 must undergo an isothermal hybridization cascade mediating formation 
 of a dicer substrate targeting y. fifth , the dicer substrate must 
 be efficiently processed by dicer to produce sirnas targeting y. sixth , 
 the scrnas should be dimensioned and/or chemically modified appropriately 
 so that only the final dicer substrate is amenable to dicer processing . furthermore , it is likely that other unanticipated design requirements 
 will emerge during the engineering and validation process ( e.g. , additional 
 constraints imposed by endogenous pathways ) . engineered scrnas that when annealed in a test 
 tube with a short rna detection target xs ( high temperature 
 followed by slow cooling to room temperature ) yielded a dicer substrate 
 that mediated knockdown of independent silencing target y upon transfection 
 into mammalian cells . engineered 
 scrnas that detect a 140-nt rna target x and produce an sirna that 
 mediates knockdown of a closely related silencing target x 
 in drosophila lysate . express an scrna in mammalian cells and transfect a short 
 modified - rna detection target xs , leading to production 
 of an sirna that mediates knockdown of independent silencing target 
 y. additional work is required to meet all six of the scrna design 
 requirements . previous research in the field of dna nanotechnology 
 demonstrates 
 that the programmable chemistry of base pairing provides a versatile 
 medium for engineering diverse dynamic functions including catalysis , 
 amplification , logic , and locomotion . we seek to exploit mechanism and sequence design principles drawn 
 from this experience to engineer scrnas ( or scdnas ) suitable for interfacing 
 with dicer and risc to mediate conditional rnai in vivo . first , we 
 must address mechanism design : how are the scrna molecules intended 
 to interact and change conformation in order to effect signal transduction ? second , we must address sequence design : given an envisioned mechanism 
 design and sequences for a pair of independent mrna detection and 
 silencing targets , x and y , what scrna sequences , if any , will encode 
 the intended signal transduction function ? to explore the mechanism 
 and sequence design challenges for conditional 
 dicer substrate formation , we have engineered five different mechanisms , 
 each satisfying the six design requirements noted above , while examining 
 diverse design alternatives spanning ( table 1 ) : reactant material ( scrna vs scdna ) , initial reactant state ( metastable 
 vs stable ) , reactant role ( catalytic vs noncatalytic ) , nucleation 
 mechanism ( toehold / toehold vs loop / toehold vs template / toehold ) , strand 
 displacement mechanism ( 3-way branch migration vs 4-way branch migration 
 vs spontaneous dissociation ) , reactant type ( hairpin monomer vs duplex 
 dimer ) , dicer substrate assembly method ( hybridization vs transcription ) , 
 and dicer substrate type ( dsirna vs shrna ) . in studying these design 
 alternatives , we sought both to optimize performance in satisfying 
 the six design requirements and to achieve simplicity . for a 
 given scrna ( or scdna ) transduction mechanism , sequence design 
 must be performed subject to the constraints imposed by a given pair 
 of mrna detection and silencing targets , x and y ( i.e. , which subsequences 
 within the full - length mrna sequences , if any , confer desirable properties 
 on the dynamic conditional response of the scrna transducers ? ) . these 
 sequence constraints dramatically reduce the size of the design space , 
 increasing the challenge of designing well - behaved sequences . here , 
 we employ nupack to solve a constrained multistate sequence design 
 problem based on a set of target secondary 
 structures representing key initial , intermediate , and final states 
 in the intended conditional hybridization cascade . sequences are optimized 
 with the goal of reducing the ensemble defect for each target structure 
 below a user - specified stop condition . for a given target secondary structure and candidate sequence , the 
 ensemble defect is the average number of incorrectly paired nucleotides 
 at equilibrium evaluated over the ensemble of ( unpseudoknotted ) secondary 
 structures . optimization of the ensemble 
 defect encompasses both a positive design paradigm ( optimize affinity 
 for the target structure ) and a negative design paradigm ( optimize 
 selectivity against all other structures in the ensemble ) . hence , multistate ensemble defect optimization provides a framework 
 for designing sequences that execute signal transduction via a prescribed 
 hybridization cascade punctuated by the desired reactant , intermediate , 
 and product secondary structures . following mechanism and sequence 
 design , we quantify the off / on 
 response of conditional dicer substrate formation in test tube studies , 
 introducing either a short rna or full - length mrna detection target , 
 and monitoring production of dicer substrates targeting an independent 
 mrna silencing target . studies with recombinant dicer are used to 
 verify that only the final product of signal transduction , and not 
 the reactants or intermediates , are efficiently processed by dicer , 
 yielding sirnas . for our engineering 
 studies , we consider detection target dsred2 
 ( mrna x ) and silencing target d2egfp ( mrna y ) . hence , our objective 
 is to design scrna mechanisms and sequences so that , upon exposure 
 to dsred2 mrna , the scrnas interact and change conformation to form 
 a dicer substrate targeting d2egfp mrna . to focus our attention on 
 scrna signal transduction and eliminate the confounding effects of 
 native mrna secondary structure , we also consider short detection 
 targets ( xs ) corresponding to the dsred2 subsequence that 
 is recognized by a given scrna mechanism . we quantify the relative 
 off / on response of conditional dicer substrate formation in the absence / presence 
 of the detection target ( xs or x ) . as a test for off - target 
 effects , we also measure the response to the silencing target y and 
 to gapdh ( mrna z ) , neither of which should initiate signal transduction . 
 to confirm that scrna transducers interact with dicer as intended , 
 we use recombinant dicer to test for undesired processing of the scrna 
 reactants and transduction intermediates as well as for efficient 
 processing of the final product ( i.e. , the cognate dicer substrate ) 
 to produce sirnas . experimental characterizations of conditional off / on 
 response ( figures 26 ) are augmented by computational and experimental stepping 
 analyses that characterize the reactants , intermediates , and products 
 for each mechanism ( sections s2s6 ) . , which has previously been used to 
 program diverse self - assembly and disassembly hybridization cascades , 
 including catalytic duplex formation . in the present circumstances , 
 the duplex that we wish to form must have the canonical 2-nt 3-overhang 
 of a dsirna , and the catalysis process must also achieve sequence 
 transduction between detection target x and silencing target y. the 
 transduction mechanism of figure 2a employs 
 three hairpins ( a , b , and c ) that coexist metastably in the absence 
 of detection target x ( i.e. , they are kinetically impeded from assembling 
 into an equilibrium distribution of products ) . the detection target 
 x opens hairpin a , which in turn opens hairpin b , which in turn opens 
 hairpin c , leading to formation of duplex bc and regeneration 
 of catalyst xa . duplex bc has a 2-nt 3-overhang 
 and targets silencing target y. chemical modifications ( 2ome - rna ) 
 of a and portions of b and c are employed to prevent dicer cleavage 
 of scrna reactants and transduction intermediates , while preserving 
 efficient dicer processing of the transduction product bc . in 
 functional terms , a detects x and catalyzes production of dsirna bc 
 targeting y. conditional catalytic dsirna formation using metastable 
 scrnas . scrna a detects mrna detection target x ( containing 
 subsequence  a - b - c - d ) to form catalyst xa , which 
 mediates production of dsirna dicer substrate bc targeting mrna 
 silencing target y ( containing independent subsequence scrnas a , b , and c coexist metastably in the absence of x. successive 
 toehold - mediated 3-way branch migrations enable assembly of x with 
 a ( step 1 ) , xa with b ( step 2 ) , xab with c ( step 
 3a ) , and disassembly of dsirna dicer substrate bc from catalyst xa ( step 3b ) . domain lengths : |a| = 10 , |b| 
 = 10 , |c| = 5 , |d| = 2 , |e| = 2 , |v| = 2 , |w| = 5 , |x| = 2 , |y| = 
 6 , |z| = 5 . chemical modifications ( 2ome - rna ) : a and parts 
 of b and c ( dashed backbone ) . : minimal production of dicer substrate bc 
 in the absence of detection target x , the presence of mrna silencing 
 target y , or the presence of mrna off - target z. on state : strong production 
 of bc in the presence of substoichiometric or stoichiometric 
 short rna detection target xs (  a - b - c - d ) 
 or the presence of full - length mrna detection target x. ( c ) quantification 
 of the dicer substrate band ( bc ) in panel ( b ) . ( d ) conditional 
 dicer processing . off state : minimal processing of the reactants ( lane 
 2 ) . on state : efficient processing of dicer substrate bc ( lane 
 4 ) , yielding canonical 21- and 23-nt sirnas ( boxed bands ) . figure 2b , c examines the conditional 
 off / on 
 response of the transduction mechanism . in the absence of the detection 
 target x , there is minimal production of dsirna bc , corresponding 
 to minimal neither the mrna silencing target 
 y ( which is necessarily related in sequence to the hairpins ) nor the 
 unrelated mrna off - target z causes measurable production of bc 
 above the background leakage . stoichiometric introduction of the short 
 detection target xs leads to strong production of bc , 
 and substoichiometric introduction of xs demonstrates catalytic 
 turnover in producing bc . strong production of bc is also 
 observed using full - length mrna detection target x. the off / on conditional 
 response of the transduction mechanism yields more than an order of 
 magnitude increase in production of dicer substrate above background 
 ( figure 2c ) . figure 2d demonstrates signal transduction 
 in the presence of recombinant dicer . only the dsirna bc that 
 is the final product of transduction is recognized and efficiently 
 processed by dicer , yielding canonical 21- and 23-nt sirnas ( see also figure s3 and table s3 ) . in assessing the 
 advantages and disadvantages of this scrna transduction 
 mechanism , it is helpful to classify the design features that contribute 
 to conditional shape change and conditional sequence change ( table 1 ) . the hairpin reactants are metastable , and could 
 potentially leak into the dsirna product on a biologically relevant 
 time scale , even in the absence of detection target x. three hairpins 
 are required to effect the necessary shape and sequence transduction : 
 hairpin a achieves partial sequence independence via the loop , hairpin 
 b moves the independent sequence to the end of the strand for presentation 
 to dicer , and hairpin c contributes the additional independent sequence 
 of the 2-nt 3-overhang and liberates the fully formed dsirna 
 bc from catalyst xa . this process requires transient formation 
 of a relatively complex intermediate ( tetramer xabc 
 including mrna x ) . use of monomer hairpins simplifies preparation 
 of purified reactants , but eventual delivery of three scrna species 
 to cells could prove burdensome . on the other hand , each self - assembly 
 operation ( opening of a new a , b , or c hairpin ) and disassembly operation 
 ( liberation of a new bc duplex ) occurs via toehold - mediated 
 3-way branch migration , providing a robust 
 framework for engineering fast reaction kinetics . the mechanism has the potentially useful property that 
 a single detection target x can catalyze production of multiple dsirnas 
 targeting silencing target y , augmenting the catalytic turnover that 
 is already present in the rnai pathway ( via risc - mediated cleavage 
 of multiple silencing targets using a single guide strand ) . we wondered 
 whether we could simplify the signal transduction mechanism 
 by exploiting alternative design principles . in particular , it seems 
 intuitively desirable to reduce the number of scrna reactants , the 
 number of assembly steps in the transduction cascade , and the complexity 
 of the reaction intermediates . these goals are achieved by replacing 
 the a and b hairpins of mechanism 1 with the ab duplex of mechanism 
 2 ( figure 3a ) . the detection target x mediates 
 displacement of a from b , which opens c to produce duplex bc 
 with a 2-nt 3-overhang . the number of reactants and the number 
 of assembly steps are both reduced from three to two and the largest 
 intermediate is reduced from a tetramer ( resulting from three sequential 
 assembly steps ) to a trimer ( resulting from one assembly step ) . this 
 simplified signal transduction mechanism dispenses with catalytic 
 turnover , producing one dsirna per detected molecule of x. in functional 
 terms , ab detects x , leading to production of dsirna bc 
 targeting y. conditional dsirna formation using stable scrnas . scrna ab detects mrna detection target x ( containing subsequence 
  a - b - c ) , leading to production of dsirna dicer substrate 
 bc targeting mrna silencing target y ( containing independent 
 subsequence  w - x - y - z ) . scrnas ab and c are stable 
 in the absence of x. a swaps b for x ( step 1 ) via toehold - mediated 
 3-way branch migration and spontaneous dissociation . b assembles with 
 c ( step 2 ) via loop / toehold nucleation and 3-way branch migration 
 to form dsirna dicer substrate bc . domain lengths : |a| = 6 , 
 |b| = 4 , |c| = 8 , |s| = 5 , |w| = 2 , |x| = 12 , |y| = 4 , |z| = 3 . chemical 
 modifications ( 2ome - rna ) : a and part of c ( dashed backbone ) . ( b ) conditional dicer substrate formation . off state : minimal production 
 of dicer substrate bc in the absence of detection target x , 
 the presence of mrna silencing target y , or the presence of mrna off - target 
 z. on state : strong production of bc in the presence of short 
 rna detection target xs (  a - b - c ) or full - length 
 mrna detection target x. ( c ) quantification of the dicer substrate 
 band ( bc ) in panel ( b ) . ( d ) conditional dicer processing . off 
 state : minimal dicer processing of the reactants ( lane 2 ) . on state : 
 efficient dicer processing of dsirna dicer substrate bc ( lane 
 4 ) , yielding canonical 2123-nt sirnas ( boxed bands ) . the mechanism exhibits strong off / on conditional dicer substrate 
 formation , achieving an order of magnitude increase in dsirna production 
 in the presence of either the short detection target xs or the full - length mrna detection target x ( figure 3b , c ) . chemical modifications of a and portions of c are employed 
 to prevent dicer processing of the reactants and intermediates . only 
 the dsirna bc is efficiently processed by dicer , yielding canonical 
 2123-nt sirnas ( figures 3d and s8 and table s5 ) . compared to mechanism 
 1 , shape and sequence transduction are achieved 
 based on dramatically altered design principles ( table 1 ) . mechanism 1 repeatedly exploits toehold / toehold hybridization 
 for nucleation and 3-way branch migration for strand displacement , 
 while mechanism 2 simplifies the transduction pathway by also exploiting 
 spontaneous dissociation to achieve strand displacement ( of b from 
 xa ) and loop / toehold hybridization to nucleate interactions 
 ( between b and c ) . strikingly , the scrnas for mechanism 2 are stable 
 rather than metastable ( i.e. , if the scrnas are allowed to equilibrate 
 in the absence of x , they will predominantly remain in the reactant 
 state rather than converting to the product state ; see section s7 ) . this is a major conceptual advantage 
 because it places a thermodynamic rather than a kinetic limit on the 
 amount of spurious dsirna that can form in the absence of x. with 
 mechanism 2 , strong production of dsirna is only thermodynamically 
 favorable if x is present , whereas with the metastable reactants of 
 mechanism 1 , x catalyzes a reaction that is kinetically impeded but 
 thermodynamically favorable in the absence of x. in our studies , the 
 metastable scrnas of mechanism 1 and the stable scrnas of mechanism 
 2 happen to produce comparable amounts of background dsirna in the 
 absence of x ( both yielding an off state that is 5% of the 
 on state achieved using xs ) . nonetheless , stable reactants 
 offer a conceptually appealing framework for engineering robust off / on 
 signal transduction in vivo ; if the thermodynamic driving force for 
 spontaneous dsirna formation can be further reduced , stable reactants 
 promise a clean and reliable off state . motivated by the simplifications of mechanism 2 , we 
 wished to see if we could push even further to simplify shape and 
 sequence transduction . mechanism 3 requires only a single duplex scrna 
 ab , and in a single step produces a dicer substrate that is 
 an shrna monomer instead of a dsirna duplex ( figure 4a ) . the detection target x mediates displacement of a from 
 b to yield a hairpin b with a 2-nt 3-overhang . the number 
 of reactants and the number of assembly steps are both reduced from 
 two to one . this is the simplest mechanism for conditional dicer substrate 
 formation that we have devised to date . in functional terms , ab 
 detects x , leading to production of shrna b targeting y. conditional 
 shrna formation using a single stable scrna . scrna ab detects mrna detection target x ( containing subsequence 
  a - b - c ) , leading to production of shrna dicer substrate 
 b targeting mrna silencing target y ( containing independent subsequence 
  y - z ) . scrna ab is stable in the absence of x. 
 x partially displaces a from b via toehold - mediated 3-way branch migration , 
 exposing a previously sequestered internal toehold , c , 
 within b , mediating a further 3-way branch migration that disassembles 
 b from xa to yield shrna dicer substrate b. domain lengths : 
 |a| = 12 , |b| = 14 , |c| = 3 , |y| = 2 , |z| = 19 . chemical modifications 
 ( 2ome - rna ) : a ( dashed backbone ) . ( b ) conditional dicer substrate 
 formation . off state : minimal production of dicer substrate b in the 
 absence of detection target x , the presence of mrna silencing target 
 y , or the presence of mrna off - target z. on state : strong production 
 of b in the presence of short rna detection target xs (  a - b - c ) 
 or full - length mrna detection target x. ( c ) quantification of the 
 dicer substrate band ( b ) in panel ( b ) . ( d ) conditional dicer processing . 
 off state : minimal processing of the reactants ( lane 2 ) . on state : 
 efficient processing of shrna dicer substrate b ( lane 4 ) , yielding 
 canonical 21- and 22-nt sirnas ( boxed bands ) . see section s4 for additional computational and experimental studies 
 of mechanism 3 . the mechanism exhibits 
 strong off / on conditional dicer substrate 
 formation , achieving 2 orders of magnitude increase in shrna production 
 in the presence of the short detection target xs and 1 
 order of magnitude increase for the full - length mrna target x ( figure 4b , c ) . it is unclear why the performance is diminished 
 for the full - length target ( though still comparable to the performance 
 for other mechanisms ) ; we expect this behavior is specific to this 
 test case and not general to the mechanism . the most striking feature 
 of these data is that the off state is undetectable ( i.e. , smaller 
 than our estimated gel quantification uncertainty ) . the clean 
 off state follows from the fact that the sirna reactant , 
 ab , is highly stable , with very little thermodynamic driving 
 force for production of shrna b in the absence of x. hence , this mechanism 
 compellingly exhibits the benefit of using stable rather than metastable 
 reactants . for mechanism 3 , x partially displaces a from b 
 via toehold - mediated 3-way branch migration , exposing a previously 
 sequestered internal toehold , which b then uses to nucleate a 3-way 
 branch migration with itself , completing displacement of a. chemical 
 modifications of a are employed to ensure that only shrna b is efficiently 
 processed by dicer , yielding canonical 21- and 22-nt sirnas ( figures 4d and s12 and table s7 ) . to date , efforts to engineer conditional 
 hybridization cascades within the field of dna nanotechnology have 
 focused almost exclusively on strand displacement reactions based 
 on 3-way branch migration , in which an invading strand displaces one 
 strand from a duplex . by comparison , 
 there has been very little study of strand displacement reactions 
 based on 4-way branch migration in which two duplexes 
 exchange partner strands . in the present setting , a dsirna signal 
 transduction product is a duplex , so we were curious if 4-way branch 
 migration might prove especially suitable for conditional dicer substrate 
 formation . mechanism 4 employs two duplex scrnas ( ab and cd 
 of figure 5a ) . the detection target x mediates 
 swapping of partner strands , producing duplex bc with a 2-nt 
 3-overhang . chemical modifications to a and d prevent dicer 
 cleavage of the reactants and intermediates , while preserving efficient 
 dicer processing of transduction product bc . in functional terms , 
 ab and cd detect x , leading to production of dsirna bc 
 targeting y. conditional dsirna formation via template - mediated 4-way 
 branch 
 migration . scrnas ab and cd detect mrna 
 detection target x ( containing subsequence  a - b - c - d - e ) , 
 leading to production of dsirna dicer substrate bc targeting 
 mrna silencing target y ( containing independent subsequence scrnas ab and cd coexist metastably in the absence of 
 x. x templates conucleation of ab and cd , mediating a short 3-way branch migration that enables toehold / toehold 
 nucleation between b and c to create a 5-way junction ( step 1a ) . subsequent 
 4-way branch migration and spontaneous dissociation disassemble dsirna 
 dicer substrate bc from xad ( step 1b ) . domain lengths : 
 |a| = 8 , |b| = 6 , |c| = 6 , |d| = 7 , |e| = 11 , |x| = 2 , |y| = 19 , |z| 
 = 2 . chemical modifications ( 2ome - rna ) : a and d ( dashed backbone ) . ( b ) conditional dicer substrate formation . off state : minimal production 
 of dicer substrate bc in the absence of detection target x , 
 the presence of mrna silencing target y , or the presence of mrna off - target 
 z. on state : strong production of bc in the presence of short 
 rna detection target xs (  a - b - c - d - e ) or 
 full - length mrna detection target x. ( c ) quantification of the dicer 
 substrate band ( bc ) in panel ( b ) . ( d ) conditional dicer processing . 
 off state : minimal processing of the reactants ( lane 2 ) . on state : 
 efficient processing of dsirna dicer substrate bc ( lane 4 ) , 
 yielding canonical 2124-nt sirnas ( boxed bands ) . additional 
 dicer products are produced by unexpected cleavage of the substrate 
 within domains  y and  y*. see section s5 for additional computational and experimental 
 studies of mechanism 4 . the mechanism exhibits strong off / on conditional dicer substrate 
 formation , achieving an order of magnitude increase in dsirna production 
 in the presence of either the short detection target xs or the full - length mrna target x ( figure 5b , c ) . only the transduction product bc is efficiently processed 
 by dicer , yielding canonical 2124-nt sirnas ( figures 5d and s17 and table s9 ) . additional dicer products are produced by unexpected cleavage 
 of the dicer substrate within domains  y and  y* , 
 suggesting that for some fraction of the substrates , dicer is either 
 measuring unusually short sirnas from the cognate end of the substrate 
 ( which has a canonical 2-nt 3-overhang ) or is measuring from 
 the noncognate end of the substrate ( which has a 7-nt 3-overhang ) . if further studies confirm the latter explanation , dicer recognition 
 of the noncognate end can be further discouraged by introducing a 
 5-overhang . this mechanism achieves sequence and shape 
 transduction using markedly 
 different design elements than mechanisms 13 ( table 1 ) . sequence transduction is achieved via the novel 
 approach of templated nucleation , with the two scrnas ab and 
 cd being brought into proximity not via mutual complementarity 
 to each other ( as with conventional toehold / toehold or loop / toehold 
 nucleation ) but due to complementarity to adjacent segments of another 
 strand  the detection target x , which serves as a template for 
 their nucleation . templated nucleation provides a simple approach 
 to sequence transduction because by construction , the template sequence 
 ( the input ) is independent from the sequences of the nucleated duplexes 
 ( the output ) . we are not aware of previous use of nucleic acid templates 
 to mediate conditional strand displacement via either 3- or 4-way 
 branch migration . after the two scrnas are colocalized via templated 
 nucleation , shape transduction is completed via 4-way branch migration 
 in which the two scrna duplexes swap base - pairing partners . initially 
 the scrnas each undergo short 3-way branch migrations with the template 
 to liberate short mutually complementary toeholds , creating a 5-way 
 junction with the template which resolves into a 4-way branch migration 
 as strand swapping commences . previous studies demonstrated that 4-way 
 branch migrations are dramatically faster when they are mediated by 
 two toehold / toehold nucleations to create an initial 4-way junction ; here we adapted this principle to the templated 
 scenario , where each duplex experiences first template / toehold nucleation 
 with x and then toehold / toehold nucleation with each other . by including 
 this auxiliary toehold / toehold nucleation step to enhance branch migration 
 kinetics , we introduced some sequence dependence of duplex bc 
 on x , which is then removed by dicer to produce a completely independent 
 sirna targeting y. template - mediated 4-way branch migration provides 
 a simple one - step approach to conditional dicer substrate formation 
 that provides an intriguing alternative to the more familiar concepts 
 of toehold / toehold nucleation and 3-way branch migration . the previous 
 mechanisms explored design alternatives for conditional 
 dicer substrate hybridization using scrnas . here , we consider the 
 alternative strategy of conditional dicer substrate transcription 
 based on signal transduction with scdnas . kim et al . have previously demonstrated conditional in vitro transcription 
 mediated by conditional hybridization of a double - stranded dna promoter 
 sequence . here , we combine conditional promoter assembly with sequence 
 transduction to implement conditional dicer substrate transcription . 
 for this test tube design study , t7 rna polymerase is employed for 
 in vitro transcription , taking advantage of well - characterized promoter 
 and termination sequences . future applications 
 in eukaryotic cells would require use of eukaryotic promoter and termination 
 sequences ( e.g. , the h1 promoter and poly - t termination sequences 
 for rna polymerase iii , which are commonly used for shrna transcription ) . mechanism 5 employs two metastable dna hairpins ( a and b of figure 6a ) . the detection target 
 x opens hairpin a , which in turn opens hairpin b via a 4-way branch 
 migration to assemble a dsdna template for transcription of rna hairpin 
 c ( including promoter , coding , and termination sequences ) . this signal 
 transduction approach incorporates the catalytic turnover inherent 
 in repeated transcription of the template . in functional terms , a 
 detects x leading to transcription of shrna c targeting y. conditional 
 shrna transcription using scdnas . scdna a detects mrna detection target x ( containing subsequence  a - b ) 
 and assembles with b to form a transcription template ( containing 
 promoter , coding , and termination sequences ) , leading to transcription 
 of the shrna dicer substrate c targeting mrna silencing target y ( containing 
 independent subsequence scdnas a and b coexist 
 metastably in the absence of x. x assembles with a via toehold - mediated 
 3-way branch migration ( step 1 ) . subsequently , xa assembles 
 with b via toehold - mediated 4-way branch migration to produce a dsdna 
 transcription template ( step 2 ) , mediating transcription of shrna 
 dicer substrate c with catalytic turnover . domain lengths : |a| = 10 , 
 |b| = 8 , |p| = 8 , |q| = 9 , |t| = 7 , |y| = 6 , |z| = 19 . ( b ) conditional 
 transcription template formation . off state : minimal production of 
 transcription template ab in the absence of short dna detection 
 target xs (  a - b ) , the presence of mrna silencing 
 target y , or the presence of mrna off - target z. on state : strong production 
 of transcription template xsab in the presence 
 of xs . ( c ) conditional dicer substrate transcription and 
 processing . off state : minimal transcription of dicer substrate c 
 in the absence of short dna detection target xs ( lane 1 ) . 
 on state : strong transcription of c in the presence of xs ( lane 3 ) and efficient dicer processing of shrna dicer substrate 
 c ( lane 4 ) . ( d ) quantification of the dicer substrate band ( c ) in 
 lanes 1 and 3 of panel ( c ) . see section s6 for additional computational and experimental studies of mechanism 
 5 . here , we characterize both the 
 off / on response of conditional transcription 
 template formation and the off / on response of conditional dicer substrate 
 transcription . for this mechanism , we engineered scdnas to detect 
 a random short dna target xs so we have not characterized 
 performance for a full - length mrna detection target x. we were nonetheless 
 able to characterize spurious transcription template formation using 
 the full - length mrna silencing target y and off - target mrna z. in the absence / presence of short dna detection target xs the mechanism demonstrates strong off / on conditional transcription 
 template formation ( figure 6b ) and transcription 
 of shrna dicer substrate c ( figure 6c ) , yielding 
 more than an order of magnitude increase in shrna production ( figure 6d ) . the transcription product c is efficiently processed 
 by dicer ( figure 6c and figure s21 ) , but the poly - u 3-overhang and short 19-bp 
 stem ( consistent with functional shrnas transcribed with rna polymerase 
 iii in vivo ) lead to noncanonical dicer products ( see section 
 s6.6 for details ) . we 
 have engineered five nucleic acid mechanisms for executing the 
 molecular logic : if mrna detection target x is present , form a dicer 
 substrate targeting independent mrna silencing target y. scrna sequences 
 encoding the desired shape and sequence transduction properties were 
 designed using the multistate sequence design feature of the nupack 
 web application , supplying mrna x ( dsred2 ) 
 and mrna y ( d2egfp ) as external sequence constraints . for each transduction mechanism , equilibrium test tube calculations were used to characterize 
 the stability of the designed reactants and to step through the intended 
 molecular assembly and disassembly operations to verify that the targets , 
 reactants , intermediates , and products were predicted to be well - formed 
 with high yield ( see section s1.8 ) . reflecting 
 the challenge of designing scrna sequences that are highly constrained 
 by the sequences of mrnas x and y , sequence domains that were intended 
 to be perfectly unstructured experiments confirmed the predicted 
 reactant stability properties , and mechanism stepping experiments 
 confirmed that the intended assembly and disassembly operations occurred 
 with high yield ( see section s1.8 ) . for each of the five mechanisms , test tube experiments demonstrated 
 a strong off / on conditional response , with at least an order of magnitude 
 increase in dicer substrate formation in the presence of the cognate 
 full - length mrna detection target x ( for the scdnas of mechanism 5 , 
 the detection target was not constrained to be an mrna sequence , so 
 only the designed short dna detection target xs was tested 
 experimentally ) . reactant structural domains were dimensioned and/or 
 chemically modified to ensure that only the cognate dicer substrates 
 that were the final products of signal transduction were efficiently 
 processed by dicer , yielding canonical sirnas for mechanisms 14 
 as well as noncanonical dicer products for mechanisms 4 and 5 . these mechanism studies explored diverse design principles for 
 shape and sequence transduction via conditional assembly and disassembly 
 of scrna and scdna complexes ( summarized in table 1 ) . in broad terms , it appears that varied design concepts 
 that have paced progress in the field of dynamic dna nanotechnology 
 ( including mechanisms for strand nucleation , strand displacement , 
 catalytic hybridization , and motif metastability ) are equally applicable to dynamic rna nanotechnology , which 
 is relatively unexplored , yet holds great potential for synthetic 
 regulation in the context of biology ; biological rnas interface with 
 diverse endogenous pathways , and hence synthetic rna signal transducers 
 that accept rna inputs and produce rna outputs represent a particularly 
 appealing framework for engineering conditional regulation in vivo . the cellular setting introduces 
 additional uncertainties beyond those addressed here , including the 
 need to deliver the scrnas and the potential for off - pathway interactions 
 ( including with endogenous pathways that are as yet undiscovered ) . the design versatility demonstrated in the present work will facilitate 
 a flexible approach in engineering around additional design constraints 
 that must be imposed going forward . if scrna - mediated conditional 
 rnai eventually performs robustly in vivo , it will provide biologists 
 with a powerful tool for the study of genetic necessity via tissue- 
 and time - specific gene knockdown . the same molecular logic would also 
 have significant medical potential , where transcript x could be chosen 
 to be a diagnostic target and transcript y could be chosen to be an 
 independent therapeutic target . more generally , if the challenges 
 of operating in vivo can be surmounted , programmable signal transduction 
 with small conditional rnas will provide an enticing conceptual framework 
 for implementing diverse modes of conditional regulation . sequences were designed and analyzed 
 using the nupack web application , and 
 oligonucleotides were synthesized by idt . duplex scrnas were purified 
 by native page prior to use . to quantify conditional off / on signal 
 transduction response , reactions were run in 100 mm potassium acetate , 
 20 mm hepes , ph 7.5 ( mechanisms 14 ) , or 50 mm na2hpo4 , 0.5 m nacl , ph 7.5 ( mechanism 5 ) at 37 c for 
 2 h ( 0.5 m per strand , with concentrations adjusted relative 
 to xs to ensure proper stoichiometry ) . dicer 
 processing was characterized using the recombinant human turbo dicer 
 enzyme kit ( genlantis ) , native page , and mass spectrometry . for mechanisms 
 14 , scrna transduction and dicer processing were performed 
 simultaneously ( 0.5 m per strand , incubated with 0.51 
 units of recombinant dicer at 37 c for 2 h ) and then characterized 
 by native page poststained with sybr gold . for mechanism 5 , scdna 
 transduction and t7 in vitro transcription were performed simultaneously 
 ( 0.1 m per strand , 37 c for 3 h in the presence of radioactive 
 utp ) . the transcription product was purified , incubated with 1 unit 
 of recombinant dicer per 20,000 cpm at 37 c for 2 h and then 
 characterized by native page visualized by phosphorimaging .
rna 
 interference ( rnai ) mediated by small interfering rnas ( sirnas ) 
 enables knockdown of a gene of choice , executing the logical operation : 
 silence gene y. the fact that the sirna is constitutively active is 
 a significant limitation , making it difficult to confine knockdown 
 to a specific locus and time . to achieve spatiotemporal control over 
 silencing , we seek to engineer small conditional rnas ( scrnas ) that 
 mediate 
 conditional rnai corresponding to the logical 
 operation : if gene x is transcribed , silence independent gene y. by 
 appropriately selecting gene x , knockdown of gene y could then be 
 restricted in a tissue- and time - specific manner . to implement the 
 logic of conditional rnai , 
 our approach is to engineer scrnas that , 
 upon binding to mrna 
 detection target x , perform shape 
 and sequence transduction to form a dicer substrate targeting independent 
 mrna  silencing target y , with subsequent dicer processing 
 yielding an sirna targeting mrna y for destruction . toward this end , 
 here we design and experimentally validate diverse scrna mechanisms 
 for conditional dicer substrate formation . 
 test tube studies demonstrate 
 strong off / on conditional response , with at least an order of magnitude 
 increase in dicer substrate production in the presence of the cognate 
 mrna detection target . by appropriately dimensioning and/or chemically 
 modifying the scrnas , only the product of signal transduction , and 
 not the reactants or intermediates , 
 is efficiently processed by dicer , 
 yielding sirnas . 
 these mechanism studies explore diverse design principles 
 for engineering scrna signal transduction cascades including reactant 
 stability vs metastability , catalytic vs noncatalytic transduction , 
 pre- vs post - transcriptional transduction , reactant and product molecularity , 
 and modes of molecular self - assembly and disassembly .