a key issue in biomedicine and biotechnology is to control processes in cells with the highest specificity without unwanted side effects of the intervening agents . gene transcription is universally controlled by structural elements within the gene , cisacting regulatory elements , which specifically bind transcription factors.1 among these cisacting elements , the core promoters bind general transcription factors of the general transcription machinery , whereas enhancer elements and proximal promoter elements bind sequencespecific transcription factors , which activate transcription by stimulating transcription initiation at the core promoter . targeting these enhancer and proximal promoter elements with , for example , synthetic sequencespecific transcription factors , represents a way to activate the transcription of only the genes harboring these elements . yet however , unlike enhancer elements , which act independently of their distance to the core promoter , the effect of proximal promoter elements depends on the distance between the transcription factor binding site and the core promoter.2 , 3 transcription factors are modular proteins composed of a dna binding domain ( dbd ) , which specifically recognizes the response element , and a transactivation domain ( tad ) , which directly or indirectly interacts with the general transcription machinery at the core promoter . therefore , if one could engineer an artificial transcription factor with a fixed distance between the dbd and the tad that matches the distance between the proximal promoter element and the core promoter of a particular gene ( figure  1 ) , one would potentially have a much more specific tool to selectively activate the transcription of this gene . a )  a dnabinding domain ( dbd ) and transactivation domain ( tad ) are separated by a rigid don of a length matching the distance between the transcription factor binding site ( tfbs ) and the core promoter of gene  a. upon binding of the dbd moiety to the tfbs , the tad would be ideally positioned to activate the transcription of gene  a. the length of the don does not match the distance between the tfbs and core promoter in gene  b. therefore , gene  b is predicted not to be activated by the construct . b )  as a test case , a rigid , tubular don ( don1 ) derivatized with the suicide ligands benzylguanine ( bg ) at one end and chlorohexane ( ch ) at the other end is functionalized with a bgreactive , snaptagged version of the dbd of the yeast gal4 transcription factor and with a chreactive , halotagged version of the vp16 tad to generate don  gv . don gv is then microinjected in zebrafish embryos together with a luciferase reporter gene construct , in which five copies of the consensus binding site for gal4 ( 5uas ) are separated from the core promoter by a spacer sequence predicted to match the length of the don . they are readily available by folding a long singlestranded dna ( ssdna ) molecule with a designed set of short synthetic  staplestrand oligonucleotides.5 , 6 , 7 dna origami nanostructures ( dons ) have typical dimensions in the 10100  nm regime , which perfectly match those of large supramolecular protein complexes , such as the molecular machinery involved in gene regulation , cell signaling , or cell division.8 more importantly , these dimensions are within the range of the typical distances between proximal promoter elements and core promoters . furthermore , dons have unique structuredirecting properties , because they can be used as scaffolds for the precise arrangement of nonnucleic acid components such as proteins or colloidal nanoparticles.9 , 10 it is , therefore , not surprising that there is increasing activity to explore dons as tools to manipulate and analyze cellular functions.11 along this line , efforts are underway to study the stability and function of dons under in  vitro and in  vivo conditions . for instance , it has been demonstrated that dons are stable reagents under cell culturing conditions.12 , 13 , 14 dons are functional as immuneactivating programmable adjuvants,15 , 16 and they can be used as drugdelivery vehicles to circumvent drug resistance.17 , 18 , 19 , 20 these studies have suggested that intracellular don uptake is usually dependent on endocytosis.18 furthermore , dons can bind to the outside of the cells to stimulate cellular transmembrane receptors.21 , 22 , 23 
  despite these exciting reports , the exploitation of dons for the analysis and manipulation of cells is still in its infancy , and there is a strong demand for further insights into the complex interactions of these synthetic biomacromolecules with the machinery of living systems . towards this goal , we report here the microinjection of a proteinfunctionalized don construct into zebrafish embryos ( figure  1 ) . our working hypothesis is that using a don to control the distance between a dbd and a tad in order to match the distance between a proximal promoter element and a core promoter would enable the selective activation of a downstream gene . as a test system , we took advantage of the gal4uas system . this binary expression system has been widely used in many model species to drive the expression of genes . the first component of the binary system consists of a synthetic transcription factor composed of the dbd of the yeast gal4 transcription factor fused to the tad of the herpes simplex vp16 protein . the second component is comprised of a synthetic gene with a tandem array of the dna binding sites for the galvp16 chimera , called the upstream activating sequence or uas ( cgg agtac t gtcct ccg),24 upstream of a minimal core promoter . this uas promoter controls the expression of downstream cloned genes with dependence on the gal4vp16 chimeric transcription factor.25 it is known that gal4vp16 fusion proteins generate robust expression from the uas responder genes in transient and stable activation assays in zebrafish.26 to allow for a quantitative assessment of transcription , we chose the firefly luciferase reporter gene as a regulated gene . to test the effect of the distance between the uas and the core promoter in the reporter plasmid , they were separated from each other by a variable spacer ( grey region of the reporter gene , figure  1 ) . the reporter plasmid was constructed by gibson cloning27 by sequentially aligning five copies of the uas , a spacer , a minimal cmv ( cytomegalie virus ) promoter and the firefly reporter gene28 ( detailed plasmid map shown in figure  s1 ) . we reasoned that , upon injection into zebrafish embryo , the level of expression of the firefly luciferase reporter should then depend on the distance between the dbd ( gal4 ) and the tad ( vp16 ) physically connected to each other through a don spacer in the supramolecular construct don to prepare the don  gv construct , we assembled a threedimensional 811148  nm rigidrod tubular don1 ( figure  2  a ) by using the singlestranded 5438  nt template 109z5 , prepared as previously described.29 
  design and analysis of dna origami nanostructures . b )  position  ( top ) and chemical structure ( bottom ) of the staple strands used for the incorporation of the suicide ligands benzylguanine  ( bg ) and chlorohexane  ( ch ) at 5  ends of the selected staple oligonucleotides . c )  ethidium bromidestained 0.75  % agarose gel of don samples before  ( not purified , np ) and after  ( purified , p ) peg purification . the supernatant ( s ) removed after precipitation contains the nonincorporated staple strands ( red arrow ) . e )  height profiles obtained from afm images along the crosssections are marked in blue and red in panel ( d ) . the boxwhisker plots show the results of the statistical analysis of 60  particles to determine their dimensions along their axes . the values for x and y were determined from the crosssections , whereas the z values were determined from the particle 's heights . to enable the selective attachment of the gal4dbd and vp16 protein at the two ends of the rod , we incorporated at the appropriate positions pairs of the smallmolecule suicide ligands benzylguanine ( bg ) and chlorohexane ( ch ) , to which snap and halotagged proteins , respectively , can be ligated.30 the bg and chmodified staple strands were prepared from nhsactivated precursors , as previously described,30 , 31 and characterized by using gel electrophoresis ( figure  s2 ) . the assembly of don1 was achieved by temperaturedependent annealing . in brief , the singlestranded dna plasmid and the staple strands at a 1:20 molar ratio were heated to 95  c and the annealing was performed by decreasing the temperature from 85 to 65  c at 1  c per cycle with each step held for 10  min and , subsequently , from 65 to 25  c at 1  c per cycle with each step held for 15  min . the assembled don1 samples were purified through polyethylene glycol precipitation32 and their integrity and correct folding was confirmed by gel electrophoresis ( figure  2  c ) , fretdependent annealing studies ( figure  s3 ) , and afm analysis ( figure  2  d ) . statistical analysis of the afm images ( figure  2  e ) revealed that the observed length ( 1465  nm ) , thickness ( 173  nm ) , and height ( 71  nm ) were in agreement with the theoretically calculated values of 148  nm length , 11  nm thickness , and 8  nm height for the rods , as depicted in figure  2  a. to synthesize the regulatory proteindecorated construct don gv ( figure  1 ) , the required proteins were produced by recombinant expression . we cloned gal4dbd cterminally fused to the snaptag through a flexible ( ggggs)3 linker ( figure  s4 ) . the corresponding protein gal4snap was overexpressed in e.  coli and purified to homogeneity by fast protein liquid chromatography ( fplc , figure  s5 ) . as native gal4 functions as a homodimer furthermore , the functionality of the novel gal4snap fusion protein to bind the uas recognition sequence was demonstrated by using a microbeadbased pulldown assay ( figure  s7 ) . the vp16 tad was cloned with the halotag fused to its nterminus ( figure  s4 ) . the corresponding halovp16 protein was overexpressed in e.  coli , purified to homogeneity by fplc , and its identity was verified with western blot analysis ( figure  s8 ) . having both regulatory proteins available as pure and bioconjugatable reagents ( figure  3  a ) to this end , a cy5labeled don1 ( don1 ) was prepared as described above and mixed with 25  molar equivalents of gal4snap and halovp16 . several controls , which included don2 , a construct identical to don1 but lacking the bg and ch ligands , and samples lacking one of the two proteins were prepared in a similar manner . the reaction products were characterized by using gel electrophoresis ( figure  3  b ) , which clearly indicated that don the experiments also indicated the absence of unspecific binding between gal4snap , halovp16 , and the don . a )  electrophoretic analysis of the conjugation of the two ch and bgmodified staple strands with the recombinant proteins halovp16 and gal4snap to be used as tad and dbd , respectively , in don  gv ( see figure  2  b ) . this is a 16  % sds gel , 20  v  cm , 1  h , stained with coomassie ; m :  page ruler prestained protein ladder . the appearance of a second band with lower electrophoretic mobility upon incubation with the ligandconjugated dna oligonucleotides ( sample  2 , 3 , 5 , and 6 ) indicates the successful conjugation of the protein with the respective staples . b )  halovp16 and gal4snap do not bind to don2 , a construct lacking ch and bgmodified staple strands ( lanes  15 ) . in contrast , don1 containing the ch and bg modifications can bind both of the proteins , as indicated from the shift of the bands in lanes  79 . note that the conjugation with halovp16 ( lane  8) induces only slight electrophoretic mobility shifts , presumably owing to net negative charge of vp16 ( pi=4.4 ) . this is a 0.75  % agarose gel , 70  v , 3  h , visualized by cy5 fluorescence imaging  ( top ) and ethidium bromide staining  ( bottom ) . we then started to analyze the behavior of the dons in  vivo in the zebrafish embryo . to this end , we used a zebrafish line stably expressing gfpfused histone h2a33 that labels the cells nuclei and , thereby , facilitates the determination of the dons subcellular localization . don1 ( approximately 500  pl , 250  nm ) was injected into the yolk of onecellstage embryos and the embryos were imaged 6  h later ( figure  4  a ) . onecell stage embryos from a zebrafish line stably expressing gfpfused histone h2a were used,33 which allows the direct localization of the cell nuclei  ( green channel ) . the cy5labeled constructs  ( red channel ) were microinjected into the yolk with don1 alone  ( a ) , the proteindecorated don ( don1gv ; b ) or don1gv along with the reporter plasmid  ( c ) . then , at 6  h postfertilization , the embryos were embedded in agarose and analyzed by using confocal fluorescence microscopy . three channels were recorded : the brightfield image  ( i ) , the signal from the gfphistone h2 in green  ( ii ) , and the cy5 signal from the dons in red  ( iii ) . the highintensity signal in the red channel shows that the large dna constructs were internalized at a high rate from the yolk . in addition , the high magnification  ( bottom images ) clearly indicates that the internalized dons are mostly cytosolic  ( orange arrow ) , with very little to no nuclear localization  ( white arrow ) . note that the presence of neither the proteins nor the reporter plasmid influenced the internalization or the subcellular distribution of the dons . scale bars :  30  m and 10  m for low and high magnification , respectively . the injected embryos survived and developed normally , suggesting that the introduced nanomaterial was well tolerated . this result shows that don1 has no obvious deleterious effects on early embryonic development and is indeed efficiently internalized from the yolk ( figure  4  a , panel  iii ) . however , comparison of the nuclei position ( gfp signal , panel  ii ) with the don fluorescent signal ( panel  iii ) indicates that the don localizes mainly to the cytosol . gv injected alone or together with the reporter plasmid ( figures  4  b and 4  c , respectively ) . the don  gv also had no obvious effect on early embryonic development and was internalized by the cells . furthermore , it was only detected in the cytosol , irrespective of the presence of the reporter plasmid ( figures  4  b and 4  c ) . gv had no effect on the expression of the reporter gene , as compared to the don1 ( data not shown ) . spherical particles with a diameter below 26  nm freely diffuse into the nucleus , whereas larger particles require a nuclear localization signal ( nls ) to actively translocate through the nuclear pore complex , with an upper size limit of about 39  nm.34 the gal4dbd used to functionalize the don  gv harbors an intrinsic nuclear localization signal.35 however , it was apparently not sufficient to drive the don  gv to the nucleus . recently , it was shown that functionalization with an nls increases the nuclear uptake of 510  nm100300  nm rodshaped polymeric gv is unlikely because of its size , but rather to a low efficiency of the gal4dbd nls in the context of the construct . stronger  nls as a peptide covalently attached to the don might increase its nuclear uptake . we report here on an ambitious concept for controlling gene transcription in a distancedependent manner in the developing zebrafish embryo , taking advantage of designed protein  dna origami nanostructures . although we could clearly demonstrate the successful synthesis of our target construct , applications in  vivo require further developments . nonetheless , we believe that our study represents an important contribution to the exploitation of dons for the analysis and manipulation of cells in  vivo . we demonstrated , for the first time , that microinjection in the yolk of the zebrafish embryo enables intracellular distribution in most of the cells of large origami constructs harboring active proteins precisely arranged in a 3d fashion . furthermore , we demonstrated that the don constructs are not toxic for the embryos and allowed normal development , at least in the early stages . we foresee that this technology could be applied to deliver complex multiprotein constructs to the cytosol and maybe also to the nuclei by means of strong nuclear localization signals . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .
abstractthe unique structuredirecting properties of dna origami nanostructures ( dons ) show great potential to specifically manipulate intracellular processes . 
 we report an innovative concept to selectively activate the transcription of a single gene in the developing zebrafish embryo . 
 we reason that engineering a designer transcription factor in which a rigid don imposes a fixed distance between the dnabinding domain ( dbd ) and the transactivation domain ( tad ) would allow the selective activation of a gene harboring the same distance between the corresponding transcription factor binding site and the core promoter . as a test case , a rigid tubular don was designed to separate the dbd of the gal4 transcription factor and the vp16 viral protein as a tad . 
 this construct was microinjected in the yolk of onecellstage zebrafish embryos , together with a reporter plasmid to assess its functionality . 
 the large don was efficiently distributed to cells of the developing embryo and showed no signs of toxicity . however , because the don showed only a cytosolic localization , it did not activate transcription of the reporter gene . 
 although this work clearly demonstrates that don microinjection enables the intracellular distribution of multiprotein architectures in most of the cells of the developing zebrafish embryo , further refinements are necessary to enable selective gene activation in  vivo .