Nanotechnology Science and Computation Part 11 doc

254 B. Samor`ı, G. Zuccheri, A. Scipioni, P. De Santis vature plots are obtained for each palindromic structure under investigation [18, 11, 8]. 4 Experimental Evidence of DNA Sequence Recognition by Mica Surface Two Crithidia segments, like those in Fig. 3, were ligated either in the tail-to- tail (Fig. 4) or in the head-to-head (Fig. 5) orientation and two palindromic dimers were constructed. Fig. 4. Pictorial representation of the tail-to-tail DNA palindromic construct. The DNA monomers are composed in a palindromic fashion and sketched as curved ribbons with directionality defined by the sequence. The two opposite DNA faces are indicated with different gray intensities. In (b), contrary to (a), the different extent of the local curvature due to the differential interaction of the two monomeric faces with the surface is taken into account. The dyadic symmetry is thus lost and the C-like shape is, more correctly, expected to be G-like, instead. In 3D, the dyad axis, which characterizes the averaged shape of the palin- dromic DNA dimers, can be oriented along any direction of space with respect to the average plane of the curved tracts. This statistical symmetry constraint also persists when the molecules are flattened on a crystal surface, such as mica in SFM images, but only two alternative directions of the dyad axis are al- lowed, parallel or perpendicular to the surface plane. In the former case both curved halves of the molecule have the same sign of the curvature sign; in the latter case the two curved halves have curvatures opposite in sign. We called these symmetry species C-like shape and S-like shape (or S*, the asterisk in- dicating the mirror image), respectively, because the curves are isomorphous with these letters (Figs. 4a, 5a). The C-like molecules will be characterized by two positive curvatures or two negative ones, depending on which end is Hierarchy in the Construction of DNA-Based Nanostructures 255 Fig. 5. Pictorial representation of the head-to-head DNA palindromic construct. See legend of Fig. 4. chosen as the starting point of the molecule (but the sequence associated will be the same, thanks to the palindromicity). On the contrary, the two main curvatures will be oppositely signed in an S-like or an S*-like shape; either with a positive followed by a negative one (S-shape), or by a negative fol- lowed by a positive one (S* shape), independent of the direction of which end is chosen as the starting point of the molecule. These two possibilities are the result of the adhesion of the three-dimensional dimeric molecules on either of their two opposite faces. In the case of C-shaped molecules (Figs. 4a, 5a), the two faces are equivalent, instead, because within either face one half exposes a sequence complementary to that of the other. We collected a large pool of SFM images (about 1500) of two palindromic DNA constructs containing the curved tract of Crithidia fasciculata, bridged head-to-head and tail-to-tail to obtain two palindromic dimers. The curvature was evaluated along all the recorded molecular profiles and averaged over all of them according to [18, 11, 8]. The average over the whole set of profiles however, did not vanish, as should be expected on the basis of equal populations of the four subclasses depicted in Figs. 4a or 5a. The two palindromic dimers exhibited the curvature profiles reported in Fig. 6: the sigmoidal shapes are similar, but oppositely signed. The non-zero curvature profiles in Fig. 6 thus monitor the imbalance of the subpopulations of the different symmetry classes. This result proved that the surface preferentially binds one of the two different faces of the curved DNA tracts (see Fig. 3) and in principle can differently modify their curvature. This is pictorially illustrated in Figs. 4b and 5b. We have seen that the two faces of the monomeric curved tracts expose either A-rich or T-rich sequences (Fig. 2). By analyzing the shape assumed by two large sets of the two palindromic dimers of the Crithidia fasciculata 256 B. Samor`ı, G. Zuccheri, A. Scipioni, P. De Santis Fig. 6. SFM ensemble average curvature profiles of the head-to-head and the tail- to-tail DNA palindromic dimers. The inversion of sequence direction results in an inversion of the sign of curvature. fragment, it turned out that the face that both dimers expose preferentially to the mica is the T-rich one [8, 17]. 5 How Effective Is This Recognition Process? In order to answer this question we must characterize the subpopulations of the different classes and compare in particular that of S with that of S*. A more precise analysis of the expected shapes for the different classes has recently been performed [10]. We have also accounted for the presence of differential interactions be- tween the two faces on each monomer and the surface. This is expected to change the symmetry of the models depicted in Figs. 4 and 5. In fact, such differential interactions can affect the curvature and flexibility, namely the in- trinsic mechanical properties of the single monomeric units within each dimer. In particular, the S and S* shapes should retain the dyad axis and the intrin- sic anti-symmetry of the curvature functions but their shapes and the related curvatures will no longer be quantitatively equivalent because of the differ- ent interactions with the surface. Thus, the two faces of G-shaped molecules being physically equivalent, they must be present on the surface in the same number but their contribution to the curvature will be equivalent to those of Hierarchy in the Construction of DNA-Based Nanostructures 257 Fig. 7. Predicted curvature profiles of the S,S*, G and G* symmetry classes of tail-to-tail and head-to-head palindromic dimers as sketched in Figs. 4b and 5b. Note the inversion of curvature signs of the two halves of the molecule when the tail-to-tail dimer is formally transformed in the head-to-head dimer. S+S* [10]. In fact, the expected curvature profiles will be as those represented in Fig. 7 for the tail-to-tail and head-to-head dimers. These predicted profiles were perfectly confirmed by those obtained experimentally (see Fig. 8) by classifying all the profiles in the four subclasses, according to their shapes, and then by averaging the curvatures plots within each subclass. The classification of the different molecules in the subclasses indicates that the T-rich face was deposited up to 12 times more frequently than the other. Therefore, the recognition effect is strong and the differential adhesion of DNA to mica not only privileges one face with respect to the other but also modifies its curvature. 258 B. Samor`ı, G. Zuccheri, A. Scipioni, P. De Santis Fig. 8. SFM ensemble average curvature profiles of the tail-to-tail palindromic DNA dimer after shape classification. The curvatures of the species are coded as in the inserted legend. 6 From Statistics to Determinism One can tailor possible applications of this recognition effect to the self- assembled integration of inorganic material in the construction of complex DNA-based nanostructures. On the other hand, this effect has been so far characterized on a statistical basis only. With the aim of building determin- istically designed nano-objects, rigid DNA structures must be brought into play. These should exhibit the same segregation of complementary bases that takes place on the two faces of a curved molecule (Fig. 2). One possibility is offered by the structures composed of multiple blocked Holliday junctions developed by Seeman [5, 12, 13, 14]. If four blocked junctions are arranged in a DNA parallelogram of the kind sketched in Fig. 9, then the shape of the resulting object is determined precisely by the size of the arms and by the number of parallelograms that are assembled together in the nano-object (for instance via sticky ends). Within each parallelogram arm, the base sequence can be made of phased A-tracts (see Fig. 8), so that all adenines will be on one side of the par- allelogram plane, while all thymines will be on the other. Since the DNA parallelogram is really a 4 nm thick object (like 2 logs of wood lying at an angle on two others) then one can design the phase of the A-tracts so that one parallelogram can lie flat on either two A-rich DNA sides, or on two T-rich Hierarchy in the Construction of DNA-Based Nanostructures 259 Fig. 9. Parallelogram with a segregation of A and T bases on its two faces. DNA sides. Studies of the interaction of these structures with flat surfaces are under way in our laboratories. References 1. S. Brown. Engineered iron oxide-adhesion mutants of the Escherichia coli phage lambda receptor. Proc. Natl. Acad. Sci. USA , 89:8651–8655, 1992. 2. S. Brown. Metal-recognition by repeating polypeptides. Nat. Biotechnol., 15:269–272, 1997. 3. H.R. Drew and A.A. Travers. DNA bending and its relation to nucleosome positioning. J. Mol. Biol., 186:773–790, 1985. 4. H.M. Keizer and R.P. Sijbesma. Hierarchical self-assembly of columnar aggre- gates. Chem. Soc. Rev., 34:226–234, 2005. 5. S. Liao, C. Mao, J.J. Birktoft, S. Shuman, and N.C. Seeman. Resolution of undistorted symmetric immobile DNA junctions by vaccinia topoisomerase I. Biochemistry, 43:1520–1531, 2004. 6. D. Rhodes and A. Klug. Helical periodicity of DNA determined by enzyme digestion. Nature, 286:573–578, 1980. 7. B. Samor`ı and G. Zuccheri. DNA codes for nanoscience. Angewandte Chemie Int. Ed., 44:1166–1181, 2005. 8. B. Sampaolese, A. Bergia, A. Scipioni, G. Zuccheri, M. Savino, B. Samor`ı, and P. De Santis. Recognition of the DNA sequence by an inorganic crystal surface. Proc. Natl. Acad. Sci. (USA), 99:13566–13570, 2002. 9. M. Sarikaya, C. Tamerler, A.K. Jen, K. Schulten, and F. Baneyx. Molecular biomimetics: nanotechnology through biology. Nature Mater., 2:577–585, 2003. 260 B. Samor`ı, G. Zuccheri, A. Scipioni, P. De Santis 10. A. Scipioni, S. Pisano, A. Bergia, M. Savino, B. Samor`ı, and P. De Santis. Sequence-dependent DNA recognition by inorganic crystal surfaces. Submitted, 2005. 11. A. Scipioni, G. Zuccheri, C. Anselmi, B. Samor`ı, and P. De Santis. Sequence- dependent DNA curvature and flexibility from scanning force microscopy im- ages. Biophys. J., 83:2408–2418, 2002. 12. N.C. Seeman. Biochemistry and structural DNA nanotechnology: an evolving symbiotic relationship. Biochemistry, 42:7259–7269, 2003. 13. R. Sha, F. Liu, H. Iwasaki, and N.C. Seeman. Parallel symmetric immobile DNA junctions as substrates for E. coli RuvC Holliday junction resolvase. Bio- chemistry, 41:10985–10993, 2002. 14. R. Sha, F. Liu, and N.C. Seeman. Atomic force microscopic measurement of the interdomain angle in symmetric Holliday junctions. Biochemistry, 41:5950–5955, 2002. 15. S.J. Sowerby, C.A. Cohn, W.M. Heckl, and N.G. Holm. Differential adsorption of nucleic acid bases: relevance to the origin of life. Proc. Natl. Acad. Sci. USA, 98:820–822, 2001. 16. S.R. Whaley, D.S. English, E.L. Hu, P.F. Barbara, and A.M. Belcher. Selec- tion of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature, 405:665–668, 2000. 17. G. Zuccheri, A. Bergia, A. Scipioni, P. De Santis, and B. Samor`ı. DNA on sur- faces: adsorption, equilibration and recognition processes from a microscopist’s view. In AIP Conference Proceedings DNA-Based Molecular Construction,Col- lege Park (MD) USA, p. 23–27, 2002. 18. G. Zuccheri, A. Scipioni, V. Cavalieri, G. Gargiulo, P. De Santis, and B. Samor`ı. Mapping the instrinsic curvature and the flexibility along the DNA chain. Proc. Natl. Acad. Sci. USA, 98:3074–3079, 2001. Adding Functionality to DNA Arrays: the Development of Semisynthetic DNA–Protein Conjugates Christof M. Niemeyer ∗ Universit¨at Dortmund Fachbereich Chemie Biologisch-Chemische Mikrostrukturtechnik Otto-Hahn Str. 6 D-44227 Dortmund, Germany christof.niemeyer@uni-dortmund.de The generation of semisynthetic DNA–protein conjugates allows one to com- bine the unique properties of DNA with an almost unlimited variety of func- tional protein components, which have been tailored by billions of years of evo- lution to specifically perform catalytic turnover, energy conversion, or translo- cation of other components. In particular, semisynthetic proteins conjugated with single-stranded DNA oligomers offer the possibility to functionalize DNA arrays with a protein content, taking advantage of the specific Watson–Crick base pairing. This chapter summarizes the current state of the art of the syn- thesis of such hybrid DNA–protein conjugates and their applications in DNA nanotechnology. The perspectives arising from this approach include the fab- rication of nanoscale elements for the sensing and transduction of biological recognition events. 1 Introduction Nature has evolved incredibly functional assemblages of proteins, nucleic acids and other (macro)molecules to perform complicated tasks that are still daunt- ing for us to try to emulate. Biologically programmed molecular recognition provides the basis of all natural systems, and the spontaneous self-assembly of the ribosome from its more than 50 individual building blocks is one of the most fascinating examples of such a process. The ribosome is a cellu- lar nanomachine, capable of synthesizing polypeptide chains using an RNA molecule as the informational template. The ribosome spontaneously self- assembles from its more than 50 individual building blocks, driven by an ∗ I wish to thank Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financially supporting our work. 262 C.M. Niemeyer assortment of low-specificity, noncovalent contacts between discrete amino acids of the protein components, interacting with distinct nucleotide bases and the phosphate backbone of the ribosomal RNA. The structures of the ribosomal subunits have recently been resolved at atomic resolution, and the atomic structures of these subunits and their complexes with two substrate analogs have revealed that the ribosome is in fact a ribozyme [26]. Our knowl- edge of the atomic structure of this complex biological nanomachine not only satisfies our desire to fundamentally understand the molecular basis of life, but it also further motivates research to emulate natural systems in order to produce artificial devices of entirely novel functionality and performance. Biological self-assembly has stimulated biomimetic “bottom-up” approa- ches to the development of artificial nanometer-scale elements, which are re- quired commercially to produce microelectronic and micromechanical devices of increasingly small dimensions in the range of ∼ 5 to 100 nm. In this re- gard, Nadrian Seeman suggested early that one should fabricate synthetic nanometer-sized elements from biomolecular building blocks [60], and nowa- days DNA is being extensively used as a construction material for the fabri- cation of nanoscale systems [61]. The simple A–T and G–C hydrogen-bonding interactions allow the convenient programming of DNA receptor moieties, which are highly specific for the complementary nucleic acid. Another very attractive feature of DNA is the great mechanical rigidity of short double helices and its comparably high physicochemical stability. Moreover, Nature provides a comprehensive toolbox of highly specific ligases, nucleases and other DNA-modifying enzymes, which can be used for processing and manipulating the DNA with atomic precision, and thus for molecular construction on the nanometer length scale. The generation of semisynthetic DNA–protein conjugates allows one to combine the unique properties of DNA with an almost unlimited variety of protein components, which have been tailored by billions of years of evo- lution to perform highly specific functions, such as catalytic turnover, en- ergy conversion, or translocation of other components. In particular, semisyn- thetic proteins conjugated with single-stranded DNA (ssDNA) oligomers, of- fer the possibility to functionalize DNA arrays with a protein content, tak- ing advantage of the specific Watson–Crick base pairing [36, 38]. This chap- ter summarizes the current state of the art of the synthesis of such hybrid DNA–protein conjugates and their application in DNA nanotechnology. This approach can be used for the self-assembly of high-affinity reagents for im- munoassays, nanoscale biosensor elements and the biomimetic “bottom-up” fabrication of nanostructured array devices. Adding Functionality to DNA Arrays 263 2 Immobilization of Proteins by Means of DNA Hybridization Protein microarrays are currently being explored as tools in proteome re- search and are highly attractive as miniaturized multianalyte immunosensors in clinical diagnostics [76, 5, 66]. The miniaturization of ligand-binding as- says not only reduces costs by decreasing reagent consumption but also leads to enhanced sensitivity in comparison with macroscopic techniques. Recent applications of protein microarrays include high-throughput gene expression and antibody screening [30], analysis of antibody–antigen interactions [8], and identification of the protein targets of small molecules [31]. Whilst DNA mi- croarrays can be easily fabricated by automated deposition techniques [44], the stepwise, robotic immobilization of multiple proteins on chemically activated surfaces is often obstructed by the instability of most biomolecules, which usually reveal a significant tendency towards denaturation. DNA-directed im- mobilization (DDI) provides a chemically mild process for the highly parallel binding of multiple delicate proteins to a solid support, using DNA microar- rays as immobilization matrices (Fig. 1a) [51, 45, 28]. Because the lateral surface structuring is carried out at the level of stable nucleic acid oligomers, the DNA microarrays can be stored almost indefinitely, functionalized with proteins of interest via DDI immediately prior to use, and subsequent to hy- bridization, regenerated by alkaline denaturation of the double-helical DNA linkers. As an additional advantage of DDI in immunoassay applications, the intermolecular binding of the target antigens by antibodies can be carried out in a homogeneous solution, instead of in a less efficient heterogeneous solid- phase immunosorption process. Subsequently, the immunocomplexes formed can be site-specifically captured on the microarray by nucleic acid hybridiza- tion [54]. The reversibility and site selectivity of DDI enables a variety of applica- tions, including the recovery and reconfiguration of biosensor surfaces, the fabrication of mixed arrays containing both nucleic acids and proteins for genome and proteome research, and the generation of miniaturized biochip elements [34]. Recent adaptations of DDI include the use of synthetic DNA analogs, namely pyranosyl–RNA oligomers, as recognition elements for the addressable immobilization of antibodies and peptides [70] and the DNA- directed immobilization of hapten groups for the immunosensing of pesticides [4]. Recently, the DDI method has been applied in functional genomics to identify the members of a small-molecule split-pool library which bind to pro- tein targets [73, 72, 17, 11, 71]. In this approach, libraries of peptide ligands are encoded by peptide–nucleic acid (PNA) tags. 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