DNA分子自组装--K

Modular construction of DNA nanotubes of tunable geometry and single-or double-stranded character

Faisal A.Aldaye,Pik Kwan Lo,Pierre Karam,Christopher K.McLaughlin,Gonzalo Cosa and Hanadi F.Sleiman *

DNA nanotubes can template the growth of nanowires 1,orient transmembrane proteins for nuclear magnetic resonance deter-mination 2,and can potentially act as stiff interconnects,tracks for molecular motors and nanoscale drug carriers 3.Current methods for the construction of DNA nanotubes result in sym-metrical and cylindrical assemblies that are entirely double-stranded 2,4–11.Here,we report a modular approach to DNA nanotube synthesis that provides access to geometrically well-de?ned triangular and square-shaped DNA nanotubes.We also construct the ?rst nanotube assemblies that can exist in double-and single-stranded forms with signi?cantly different stiffness.This approach allows for parameters such as geometry,stiffness,and single-or double-stranded charac-ter to be ?ne-tuned,and could enable the creation of designer nanotubes for a range of applications,including the growth of nanowires of controlled shape,the loading and release of cargo,and the real-time modulation of stiffness and persist-ence length within DNA interconnects.

At present,DNA nanotubes are synthesized by vertically aligning DNA duplexes into a curved motif,followed by its closure 4–11,or by rolling and cyclizing a two-dimensional DNA origami array 2.The method described here involves the initial construction of geometri-cally well-de?ned single-stranded and cyclic DNA building-blocks with rigid organic vertices,such as the DNA triangle 3and square 4in Fig.1.These units are then longitudinally assembled via linking strands to form ‘rungs’,producing nanotubes of pre-designed architectures (Fig.2).The structure of these rungs is what ultimately dictates the ?nal geometry of the nanotubes being constructed,so creating an opportunity to control and modulate the shape and size of each nanotube,one rung at a time.Our group recently developed a class of cyclic and single-stranded DNA building-blocks that are ideal candidates for structural scaffolding 12–14.These building-blocks contain well-de?ned syn-thetic molecules as their corner units.They have been used in DNA nanotechnology to generate nanoparticle assemblies that are structurally addressable in real time,and discrete three-dimensional DNA cages capable of oscillating between several prede?ned dimen-sions.Here,we use single-stranded and cyclic triangle 3and square 4(Fig.1)to generate geometrically well-de?ned triangular and square DNA nanotubes.

To construct a triangular DNA nanotube 3nt ,for example,a single-stranded triangular template 3is used as a scaffold to ?rst generate a well-de?ned triangular building-block 30(Fig.2a).This is achieved by hybridizing 3to three complementary DNA strands (CS )that contain sticky-end overhang cohesions to form 3–3,followed by the addition of three rigidifying strands (RS )to spatially orient each of these sticky-end overhangs above and

below the plane of triangle 30.Three double-stranded linking strands (LS )of appropriate sequence then assemble the set of build-ing-blocks 30into the well-de?ned triangular DNA nanotubes 3nt (Fig.2b).Considering the ease with which geometrically unique cyclic and single-stranded DNA templates—such as triangles,squares,pentagons and hexagons—have been previously syn-thesized in our laboratory 15,this approach can be used to generate nanotubes of any arbitrary shape and size.This method enabled the synthesis of square DNA nanotubes 4nt from the cyclic and single-stranded square template 4(Fig.1),using the well-de?ned square rung 40and the double-stranded linking strands dsLS 0(Fig.2).Initial efforts to make use of this new method focused on constructing 30and 40from triangle 3and square 4,respectively.Accessing the DNA building-blocks 3and 4?rst involves synthesiz-ing a single continuous DNA strand,embedded with the appropri-ate number of vertex 1molecules (that is,three for 3and four for 4).This is followed by its subsequent cyclization using a template strand of DNA,and its chemical ligation using cyanogen bromide (see Supplementary Fig.S3).Digestion assays using ExoVII con?rm the cyclic and single-stranded nature of 3and 4(see Supplementary Fig.S4).The assembly of the triangular rung 30from one unit of template 3,three complementary strands with sticky-end overhangs,and from three rigidifying strands,is moni-tored sequentially using native polyacrylamide gel electrophoresis (PAGE).This process is found to occur quantitatively at every step leading to and including 30(see Supplementary Fig.S5).Square scaffold unit 40is assembled similarly from template 4(see Supplementary Fig.S5).

With units 30and 40in hand,we proceeded to examine their potential to generate well-de?ned triangular 3nt and square 4nt DNA nanotubes.A hierarchical approach was deemed necessary here to ensure the overall ?delity of the assembly process.In the case of 30,for example,one of the sides is programmed to contain sticky-end overhangs that are seven bases long,and the remaining two sides contain overhangs that are only ?ve bases long.Therefore,as the mixture slowly cools,the longer sticky-ends cohere ?rst to generate linear assemblies of 30molecules,followed by the favourable cohesion of the remaining pre-organized,

1

3

4

Figure 1|Single-stranded and cyclic DNA templates 3and 4.

Department of Chemistry,McGill University,801Sherbrooke Street West,Montreal,Quebec H3A 2K6,

Canada.*e-mail:hanadi.sleiman@mcgill.ca

shorter sides.This design feature allows a high synthetic yield for constructing the ?nal triangular DNA nanotubes 3nt .An analysis of the resulting assemblies using atomic force microscopy (AFM)and confocal ?uorescence microscopy reveals the formation of well-de?ned DNA nanotubes extending over several micrometres (Fig.3;see also Supplementary Information).

Lateral cross-sectional analysis conducted on the triangular nanotubes 3nt show them all to be of the same height (see Fig.3a;see also Supplementary Fig.S6b).This is consistent with a well-de?ned DNA nanotube assembly of a single size.Longitudinal analysis was conducted on the rungs within each nanotube.To do so,we modi?ed each rung with protruding hair-pins to provide a visual probe detectable using AFM.Hairpins were introduced into each corner unit of 30,to generate 30–hp,so that upon assembly,a triangular nanotube 3nt–hp with hairpins radially protruding from each of its rungs was generated (Fig.3b).The distance between each rung is estimated to be 15nm.A uniform one-dimensional array of hairpins with a periodicity of 45nm (exactly three times the inter-rung distance of 15nm)is observed,as con?rmed by Fourier analysis of the height trace (see Fig.3b;see also Supplementary Fig.S10).This is consistent with a 408rotation of each triangular rung with respect to the next one,and a realignment of the hairpins at every fourth rung (Fig.3c).Thus,the triangular nanotube assembly 3nt–hp probably possesses a helical screw axis,with nine rungs for each full turn (see Supplementary Fig.S11).Our approach to constructing DNA nano-tubes therefore generates geometrically well-de?ned DNA nano-tubes of excellent uniformity laterally and longitudinally.

DNA nanotubes that are laterally constructed one rung at a time can be easily modulated with respect to size and shape.Thus,in addition to triangular DNA nanotubes 3nt ,geometrically well-de?ned square DNA nanotubes can be readily achieved by starting with the square DNA template 4(Fig.1).Assembly of 4into 40(as with 30above)equips this template with cohesive DNA strands above and below its plane (Fig.2).Addition of

double-stranded linking strands dsLS 0generates square DNA nanotubes 4nt (Fig.4a).AFM analysis con?rms the construction of highly uniform nanotubes with extension over several micro-metres,and shows them to be of uniform size (see Supplementary Figs S7and S8).In principle,this approach can be seamlessly adapted to construct a new class of designer DNA nanotubes with rungs of different sizes and shapes,with unique sets of structural and functional properties.

DNA nanotubes that can either be completely double-stranded and rigid,or single-stranded and more ?exible,can also be con-structed using our approach.If the linking strands that join the square rungs 40are double-stranded,then fully double-stranded well-de?ned DNA nanotubes 4nt are obtained,as described above.However,if three of the four linking strands used within this system are single-stranded,then DNA nanotubes 4nt–ss with single-stranded regions are generated (Fig.2).These nanotubes now possess one double-stranded side and three single-stranded sides,and are thus expected to be less stiff.When characterized by AFM analysis,4nt–ss nanotube assemblies are found to be sig-ni?cantly more ?exible,with relatively shorter distances between regions of bending,when compared with their fully double-stranded analogues 4nt (see Supplementary Fig.S12).To our knowledge,this is the ?rst example of a DNA single-stranded nanotube.This approach thus allows deliberate control of stiffness and persistence length.It also opens the door to the possibility of using these nanotubes in their more accessible single-stranded form to allow loading of materials such as biomolecules or drugs,and subsequently closing them to their fully double-stranded form to ensure encapsulation.

In summary,we have shown a modular approach to DNA nano-tube construction that offers new elements of structural control in the assembly process of these materials.Geometry,size,stiffness,and double-or single-stranded character can all be tuned and modi?ed,thus providing ready access to deliberately designed nanotube archi-tectures.The method uses single-stranded and cyclic DNA

templates

3

CS

3-3

3'

3dsLS

4nt

4nt-ss

3nt

Figure 2|Construction of DNA nanotubes.a –c ,Construction of triangular and square rungs 30and 40(a ),triangular nanotubes 3nt (b ),square nanotubes 4nt and single-stranded square DNA nanotube 4nt –ss (c ).

3-3

3

3'-hp

i

ii

iii

iv

3nt-hp

RS-hp

3nt

45 nm

45 nm

Figure 3|AFM characterization of triangular DNA nanotubes.a ,The construction of DNA nanotubes 3nt from triangle 30results in well-de?ned one-dimensional DNA assemblies that extend over several micrometres,which from cross-sectional analysis (lower half of ?gure)are shown to be of the same diameter.Scale bar,2.5m m.b ,Hairpins incorporated into the corner units of each triangular rung 30–hp using rigidifying strands RS–hp assemble into triangular DNA nanotubes 3nt–hp with radially protruding hairpins from each of the corner units.Cross-sectional analysis (lower half of ?gure)reveals a spacing between each consecutive hairpin of 45nm (exactly three times the distance between two consecutive rungs)in which each rung is rotated by an angle of 408.Scale bar,1m m.c ,T o better illustrate the helicity within 3nt–hp ,a top-view sectional analysis is shown.(i)A single hypothetical hairpin is selected within rung 1.(ii)Superimposition of rung 2,separated from rung 1by a distance of 15nm and rotated by an angle of 408,does not result in the alignment of any of its hairpins onto any of those within rung 1.(iii)Rung 3,now separated from rung 1by a distance of 30nm and rotated with respect to rung 1by an angle of 808,still does not align.(iv)Rung 4,however,which is now separated from rung 1by a distance of 45nm and is rotated with respect to rung 1by an

angle of 1208,is superimposed onto the hairpins being monitored within rung 1,and thus results in the experimentally observed periodicity of 45nm.

4nt

4nt-SS

Figure 4|AFM characterization of square DNA nanotubes that can be double-or single-stranded.a ,b ,The same well-de?ned square rung 40is used to generate fully double-stranded square DNA nanotube 4nt assemblies using four double-stranded linking strands (a )and partially single-stranded DNA

nanotube 4nt–ss assemblies using one double-stranded linking strand and three single-stranded linking strands,which cross-sectional analysis (lower half of ?gure)shows to be of a uniform size (b ).Scale bar,2.5m m.

to generate‘rungs’of well-de?ned geometries,and assembles these units longitudinally to produce nanotubes with pre-designed struc-tures.Triangular and square DNA nanotubes can be readily accessed in their single-stranded and double-stranded forms.These assemblies are highly uniform,both longitudinally and laterally.Numerous applications for these nanostructures can be envisaged,such as the growth of metallic or semiconductor nanowires of tunable size and geometry,encapsulation of proteins or nanoparticles in their double-stranded form and possible release in their single-stranded form,and as interconnects with switchable persistence lengths. Received4November2008;accepted5March2009;

published online12April2009

References

1.Yan,H.,Park,S.H.,Finkelstein,G.,Reif,J.H.&LaBean,T.H.DNA-templated

self-assembly of protein arrays and highly conductive nanowires.Science301, 1882–1884(2003).

2.Douglas,S.M.,Chou,J.J.&Shih,W.M.DNA-nanotube-induced alignment of

membrane proteins for NMR structure determination.Proc.Natl https://www.wendangku.net/doc/4a14948311.html,A 104,6644–6648(2007).

3.Geng,Y.et al.Shape effects of?laments versus spherical particles in?ow and

drug delivery.Nature Nanotech.2,249–255(2007).

4.Rothemund,P.W.K.et al.Design and characterization of programmable DNA

nanotubes.J.Am.Chem.Soc.126,16344–16352(2004).

5.Mitchell,J.C.,Harris,J.R.,Malo,J.,Bath,J.&Turber?eld,A.J.Self-assembly of

chiral DNA nanotubes.J.Am.Chem.Soc.126,16342–16343(2004).

6.Sherman,W.B.&Seeman,N.C.Design of minimally strained nucleic acid

nanotubes.Biophys.J.90,4546–4557(2006).

7.Endo,M.,Seeman,C.&Majima,T.DNA tube structures controlled by a four-

way-branched DNA connector.Angew.Chem.Int.Ed.44,6074–6077(2005).

8.Park,S.H.et al.Three-helix bundle DNA tiles self-assemble into2D lattice or

1D templates for silver nanowires.Nano Lett.5,693–696(2005)

9.Liu,D.,Park,S.H.,Reif,J.H.&LaBean,T.H.DNA nanotubes self-assembled

from triple-crossover tiles as templates for conductive nanowires.Proc.Natl https://www.wendangku.net/doc/4a14948311.html,A101,717–722(2004).10.Mathieu,F.et al.Six-helix bundles designed from DNA.Nano Lett.5,

661–665(2005).

11.Kuzuya,A.,Wang,R.,Sha,R.&Seeman,N.C.Six-helix and eight-helix DNA

nanotubes assembled from half-tubes.Nano Lett.7,1757–1763(2007).

12.Aldaye,F.A.&Sleiman,H.F.Sequential self-assembly of a DNA hexagon as a

template for the organization of gold nanoparticles.Angew.Chem.Int.Ed.118, 2204–2209(2006).

13.Aldaye,F.A.&Sleiman,H.F.Dynamic DNA templates for discrete gold

nanoparticle assemblies:Control of geometry,modularity,write/erase and structural switching.J.Am.Chem.Soc.129,4130–4131(2007).

14.Aldaye,F.A.&Sleiman,H.F.Modular access to structurally switchable3D

discrete DNA assemblies.J.Am.Chem.Soc.129,13376–13377(2007).

15.Aldaye,F.A.,Palmer,A.L.&Sleiman,H.F.Assembling materials with DNA as

the guide.Science321,1795–1799(2008).

Acknowledgements

The authors would like to thank the Natural Sciences and Engineering Research Council of Canada,the Canada Foundation for Innovation,the Centre for Self-Assembled Chemical Structures,and the Canadian Institute for Advanced Research for?nancial support,

A.L.Palmer for help in manuscript preparation,and J.Hedberg for help in preparing the graphical illustrations.F.A.A is a McGill University Principal’s Prize Fellow.P.K.L.and P.K.thank CIHR for a Chemical Biology Scholarship.H.F.S is a Cottrell Scholar of the Research Corporation.

Author contributions

All authors discussed the results and commented on the manuscript.H.F.S.conceived and designed the project,analysed the data and co-wrote the paper.F.A.A conceived and designed the project,performed the experiments,analysed the data and co-wrote the paper. P.K.L performed the experiments and analysed the data.P.K.and G.C.performed the confocal?uorescence microscopy measurements.C.K.M.assisted in project design. Additional information

Supplementary information accompanies this paper at https://www.wendangku.net/doc/4a14948311.html,/ naturenanotechnology.Reprints and permission information is available online at http://npg. https://www.wendangku.net/doc/4a14948311.html,/reprintsandpermissions/.Correspondence and requests for materials should be addressed to H.F.S.