we first tested the proposed strategy by designing a symmetric rna  dna hybrid dx motif ( fig .
, there are two parallel hetero - duplexes that are joined together at two points where strands cross over from one duplex to the other .
the dx motif is composed of five nucleic acid strands : one central , long , two times repetitive dna strand ( l2 ) , two identical , short , peripheral dna strands ( s ) and two rna strands ( r ) .
although strands l and s cross between duplexes , each r strand continuously extends through one duplex from one end to the other . a twofold rotational axis passes through the centre of the motif and is perpendicular to the tile plane . because of this symmetry , the two s strands are identical , as are the two r strands .
each dx tile only needs three unique , component strands : l , r and s. strand r is an rna molecule , and strands l and s are both dna molecules .
the dx tiles have single - stranded overhangs ( sticky ends ) at each end of the two duplexes .
two neighbouring dx tiles can then associate with each other through hybridization of these sticky ends .
the distance between the two crossover points in the tile is set as two turns ( 22 bp ) . to allow the dx tiles to tessellate a plane instead of forming one - dimensional arrays , it is essential to keep any two adjacent
, interacting dx tiles coplanar , which requires the distance between the two adjacent crossover points of two interacting dx tiles to be n + 0.5 turns ( n is an integer ) . in the current design
the self - assembly of rna  dna hybrid nanostructures is a one - pot process .
an aqueous - solution mixture of the component dna and rna strands at the designated ratio is slowly cooled from 70 c to 25 c over 24 h. in the process , the dna  rna strands recognize and associate with each other , first into individual dx tiles and then into large assemblies .
the assemblies were analysed by native polyacrylamide gel electrophoresis ( page ) and visualized by atomic force microscopy ( afm ) . in native page ( supplementary fig .
s2 ) , all dna  rna complexes appeared as sharp bands with the expected mobility , indicating that the hybrid complexes were formed and stable .
large two - dimensional crystals and tubular structures were clearly visible ( fig . 1 ; supplementary fig .
the apparent height of the single - layered two - dimensional crystals was 2.1 nm , a reasonable value for the diameter ( 2.5 nm ) of a - form duplexes as rna 
the two - dimensional crystals showed clear periodicities both along and perpendicular to their helical axes .
the distance between two adjacent crossover points of two interacting dx tiles ( 2.5 turns or 29 bp , as indicated in fig .
1b , inset ) is important for ensuring that all dx tiles are coplanar in the final assemblies .
if this distance deviates from this value , the helical nature of the duplexes would cause any two interacting tiles to be on different planes , and promote the formation of tubular structures .
when this distance is decreased by 1 or 2 bp from 29 bp , the hybrid dx tiles are still able to assemble into large structures , which are predominantly tubular ( their heights are much higher than 2.5 nm , the diameter of an a - form duplex ) , and sometimes extended two - dimensional crystals . the tubular structures are occasionally ripped into monolayers , the regular patterns of which can be observed .
when the distance increases by 1 bp or decreases by 3 bp , the hybrid dx tiles do not form any large , regular assemblies .
overall , the formation of tubules is preferred over two - dimensional arrays in all cases , and rna  dna hybrid dx tiles are more prone than pure dna dx tiles to form tubular structures .
the strategy of using dna to program rna self - assembly is a general approach and can be used for the construction of a variety of structures . to demonstrate this capability , we engineered two rna  dna hybrid star motifs .
symmetric dna star motifs are a family of related nanostructures that have different numbers of branches ( 3 , 4 , 5 and 6 ) .
each motif is assembled from three different strands : a long repetitive strand ( l ) , a medium strand ( m ) and a short peripheral strand ( s ) .
all star motifs share the same strands m and s , but differ from one another in strand l. the sequence repeating time of the l strand determines the branch number of the motif . in the current work ,
the m strand is replaced by an rna strand ( r ) , resulting in rna  dna hybrid star motifs .
rna residues comprise half of the nanomotifs . when appropriate sticky ends are present at the peripheral ends of the duplexes , the hybrid motifs can further associate with each other into two - dimensional arrays ( figs 2 and 3 ) .
the l strands contain unpaired , single - stranded loops ( shown in orange in figs 2 and 3 ; three or four bases long for the three- or four - pointed star , respectively ) at the centre .
the short loops are long enough to prevent the branches from stacking with one another at the centre , and short enough to prevent the tiles from being too flexible .
to prevent the intrinsic curvatures of the tiles from accumulating in one direction , a corrugation strategy was also applied12,33,34 .
any two interacting tiles are separated by 4.5 turns , so the two adjacent tiles are related by a twofold rotational axis that is perpendicular to the molecular plane .
this arrangement cancels the intrinsic curvatures of the tiles and promotes the tiles to form extended two - dimensional arrays . during slow cooling , the rna 
dna hybrid star tiles readily assemble into two - dimensional crystals , which can be visualized by means of afm imaging .
the four - point star tiles assemble into periodic two - dimensional arrays ( fig .
2 ) with an apparent height of 2.2 nm . the cavities in the resulting two - dimensional crystals , to our surprise , are not squares . instead
their dimensions are ( 27.2  1.6 nm )  ( 19  1.4 nm ) .
this observation suggests that the overall geometry of the individual rna  dna hybrid tile is a skewed four - point star or a four - leafed pinwheel ( fig .
any two opposite leafs of the hybrid star are shifted away by 4 nm from one another in the direction perpendicular to the helical axes .
the overall tile structure is quite different from that of its homo - dna counterpart .
it is unlikely that this is due to a surface phenomenon of differential adsorption onto the mica substrate .
the two faces of the two - dimensional arrays are identical to each other , as many twofold rotational symmetry axes ( for example , the twofold rotational axes indicated by the bold black arrows shown in figs 2b and 3b ) exist in the plane . a similar phenomenon , although less obvious , can be observed for the rna  dna hybrid three - point star motif ( fig .
its overall geometry is a skewed three - point star or three - leafed pinwheel with a threefold rotational symmetry .
consequently , two - dimensional arrays of such tiles only have threefold , instead of sixfold , rotational symmetries .
the strategy of dna - programmed rna self - assembly is not limited to two - dimensional self - assembly ; it can also be applied to three - dimensional self - assembly . to demonstrate the three - dimensional assembly
each vertex has a connectivity of 3 and can be represented by a three - point star tile .
the hybrid three - point star is similar to the one used for assembling trigonal two - dimensional arrays .
however , the three - point star tiles in dodecahedra are significantly bent away from a planar structure . to provide enough flexibility for the tile to bend , the central single - stranded loops on the l strand are elongated from 3 to 11 bases . in addition
, any two interacting tiles are designed to be separated by four turns ( an integral number of turns ) . at such a separation
, any two interacting tiles will face the same side of the tile plane , and any intrinsic tile curvatures will accumulate in the same direction . in turn , this will promote the tiles to assemble into closed , discrete structures17 . similarly to two - dimensional self - assembly , the assembly process for three - dimensional structures is a simple one - pot process .
dna hybrid dodecahedra have been characterized by dynamic light scattering ( dls ) and cryogenic electron microscopy ( cryoem ) imaging .
the hydrodynamic radius of the hybrid complex is 20.7  1.3 nm as measured by dls .
the value is consistent with the radius ( 20.5 nm ) of the circumscribed sphere of the hybrid dodecahedron model , assuming a pitch of 0.26 nm / bp and a diameter of 2.3 nm for the rna 
4 ) . in the raw images , randomly distributed dodecahedral particles are observed , which are of the expected size . some small fragments and
large aggregates also exist  it is not clear whether those undesired structures are formed during the assembly process or result from damage accompanying sample preparation .
the yield of the correctly formed particle is 40% , based on visual observation of the cryoem images ( this is significantly lower than the assembly yield of pure dna dodecahedrons ) . from the experimentally observed particles , a three - dimensional map of the rna 
dna hybrid dodecahedron is generated by single - particle three - dimensional reconstruction , a powerful technique routinely used by structural virologists35 .
the map resolution was determined to be at 2.5 nm using the fourier shell correlation ( 0.5 threshold criterion ) of two three - dimensional maps independently built from half data sets .
there is clear similarity between the computer - generated two - dimensional projections from the reconstructed structural model and the raw , individual particle images ( fig .
4e ) , and the class averages of raw particle images with similar views ( supplementary fig .
the radius of the reconstructed dodecahedron model is 20.5 nm , consistent with both the design and the dls data .
the skewness of the star motif is not observed from the reconstructed structural model , as it was in the two - dimensional arrays , presumably due to the low resolution of our cryoem data .
when considering which strand of the overall assembly should be rna , our guideline was to make sure that every helical domain was composed of one dna strand and one rna strand .
this ensures that all helical domains will be uniform , hybrid a - form duplexes .
red strands in the tiles , but not for the green or black strands ( according to the colour code of the figures ) . theoretically , it is also possible to simultaneously use rna for the green and black strands and dna only for the red strand .
it is also possible to use rna only for all three strands ( green , red and black ) .
however , we have not experimentally explored this possibility , and it is not our intention , either . in general ,
rna strands are more difficult to prepare , much less stable , and much more expensive than dna strands with exactly the same sequence .
thus , we decided to use the minimum number ( here only one ) of rna strands in the current study . for two - dimensional self - assembly of star motifs of either homo - dna or rna  dna hybrid ,
the symmetry remains the same : three- or fourfold rotational symmetry ( figs 2 and 3 ) . in the design of the star motifs ,
each branch is an identical crossover structure and three- or fourfold rotational symmetries are expected . in the previous homo - dna structures , the star motif exhibits near d3 or d4 symmetry .
those higher - degree symmetries are a surprising finding , and not the results expected from the design .
dna structures is likely due to the subtle difference between a- and b - form duplexes at the central region of the tile ( supplementary fig .
the base pairs stack directly onto one another and go through the centre of the dna helix .
in contrast , base pairs in an a - form duplex are shifted from one another , and a cavity appears in the centre of the duplex when looking down the duplex .
in addition , the negative charges on the phosphate groups are distributed differently along the different forms of the duplexes .
electrostatic interactions would affect the homo- or hybrid duplexes differently , causing a structural adjustment to minimize the electrostatic repulsion .
single - stranded loops ( 11 bases long ; the orange segments in strand l3 ) were used at the centre to provide enough flexibility for the motif to bend , but a dodecahedron formed instead of a tetrahedron .
dna hybrid star motifs are more rigid than their corresponding homo - dna star motifs .
the most important challenge for rna self - assembly is to rationally design and construct well - defined , stiff , branching motifs , the basic building blocks for nanoconstruction . to appreciate the level of challenge
, a four - arm dna junction always adopts a right - handed , x - shaped structure .
dna hybrid junction can be either right- or left - handed36,37 . compared with this challenge ,
it is simple and straightforward to adjust the repeating length for the different duplex forms . in the present study ,
we have also observed numerous different behaviours of the a- and b - form duplexes in nanoconstruction , including the strong tendency of hybrid dx to form tubular structures , the skewed appearance of the star motifs , and the surprising three - dimensional assembly results .
star motifs ( with four - base - long sticky ends ) associate with one another and form low - mobility , high - molecular - weight species ( smears ) in the absence of divalent cations ( supplementary fig .
under the same conditions , the corresponding homo - dna motifs will not associate with each other .
dna hybrid structures in the current study were chemically less stable than the corresponding homo - dna structures .
for example , homo - dna structures do not change noticeably under afm imaging after being stored for one week at 4 c , but the rna 
dna hybrid structures will degrade , and no well - defined nanoarray can be found under afm imaging after such storage . as well as the current approach for introducing rna into dna nanostructures , a more straightforward method is simply to create dna nanostructures with appended , single - stranded oligonucleotides with which rna strands can hybridize .
furthermore , the rna moieties will be fully accessible to the surrounding solvent , harsh chemicals and degrading enzymes ( rnases ) , and are therefore prone to degradation . with the strategy reported here ,
it is possible to tightly fold rna deep inside the nanostructures to prevent such rnase accessibility ( this is in our study plan ) . in summary
the presented strategy of using dna to program rna self - assembly comprises a versatile method for assembling rna molecules into a range of nanostructures .
we believe that this strategy can be applied to all established dna nanomotifs and dna origami . some unexpected phenomena ( such as the fact that the star motifs are not straight ) have also emerged . to fully understand such phenomena and achieve tight structural controls , a systematic , theoretical modelling and experimental studies are necessary .
we expect this strategy to provide a unified platform for assembling both rna and dna into large nanostructures with multifunctional rna modalities : sirnas , micrornas , antisense rnas , ribozymes and aptamers .
dna nanoparticles for cellular deliveries of small rnas : aptamers for cellular targeting and other small rna 
rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands
were combined according to the correct molecular ratios in a tris  acetic  edta 
mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature .
after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna  dna sample solutions
were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen .
data were recorded using a gatan 4k  4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope .
three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra
was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images .
rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands
were combined according to the correct molecular ratios in a tris  acetic  edta 
mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature .
after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna  dna sample solutions
were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen .
data were recorded using a gatan 4k  4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope .
three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra
was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images .
 dna has recently been used as a programmable  smart building block for the assembly of a wide range of nanostructures . 
 it remains difficult , however , to construct dna assemblies that are also functional . 
 incorporating rna is a promising strategy to circumvent this issue as rna is structurally related to dna but exhibits rich chemical , structural and functional diversities . 
 however , only a few examples of rationally designed rna structures have been reported . 
 herein , we describe a simple , general strategy for the de novo design of nanostructures in which the self - assembly of rna strands is programmed by dna strands . to demonstrate the versatility of this approach , we have designed and constructed three different rna  
 dna hybrid branched nanomotifs ( tiles ) , which readily assemble into one - dimensional nanofibres , extended two - dimensional arrays and a discrete three - dimensional object . 
 the current strategy could enable the integration of the precise programmability of dna with the rich functionality of rna . 
Results
Discussion
Conclusions
Methods
Oligonucleotides, formation of RNADNA complexes and imaging
Single particle reconstruction
Supplementary Material