Designed Dendrimer Syntheses by Self-Assembly of Single-Site

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NANO LETTERS

Designed Dendrimer Syntheses by Self-Assembly of Single-Site, ssDNA Functionalized Dendrons

2004 Vol. 4, No. 5 771-777

Cordell R. DeMattei,† Baohua Huang,† and Donald A. Tomalia*,†,‡ Dendritic NanoTechnologies, Inc., Central Michigan UniVersity, 2625 Denison DriVe, Mt. Pleasant, Michigan 48858 Received October 30, 2003; Revised Manuscript Received March 14, 2004

ABSTRACT Single site, functionalized, single stranded (ssDNA) dendri-poly(amidoamine) (PAMAM) di-dendrons have been synthesized by covalently conjugating complementary 32 base pair oligonucleotides to single-site, thiol functionalized dendri-PAMAM di-dendrons possessing neutral or anionic surface groups. Combining these complementary (ss-DNA) functionalized PAMAM di-dendrons at appropriate assembly temperatures produced Watson−Crick base paired (dsDNA) cores, surrounded by four PAMAM dendrons. These novel core−shell nanostructures represent a new class of precise monodisperse, linear-dendritic architectural copolymers. Using comparative gel electrophoresis, it was demonstrated that these self-assembled (di-dendron) dendrimers could be hemispherically differentiated as a function of surface chemistry as well as generational size. This new supramacromolecular approach offers a very facile and versatile strategy for the combinatorial design of size, shape, and surface substituents for both homogeneous and differentiated dendritic nanostructures.

Since the initial creation of dendrimers in the early 1980s, these precise,1,2 core-shell constructs have become widely accepted as perhaps the most important members of the recently recognized new architectural class of macromolecules known as dendritic polymers.3 This extraordinary interest is due to the fact that dendrimers represent a broad range of organic/organometallic compositions and architectures that may be structure controlled as a function of (a) size, (b) shape, (c) flexibility, and (d) surface chemistry in the nanoscale region.4 It is from this perspective that dendrimers are viewed as fundamental, nanometer-sized building blocks5,6 that enable the construction of a wide range of nanoscale complexity/ devices exhibiting important uses and properties in the (a) biomedical,5,7,8 (b) nanoelectronics, (c) advanced materials,9,10 and (d) nanocatalyst fields, to mention a few.5,11,12 Covalent construction of dendrimers by the assembly of reactive monomers,13 branch cells,2,11,14 or dendrons12 around atomic9,10 or molecular cores with adherence to either divergent or convergent, dendritic branching principles is now well documented.1,15 Such a systematic occupation of nanospace around cores with monomers or branch cells, as a function of generational growth stages (i.e., monomer shells), to give discrete, quantized bundles of mass has been well demonstrated. These parameters have been shown to be mathematically predictable6,16 and exhaustively confirmed * Corresponding author. Phone: 989-774-3096, Fax: 989-774-2322, Email: [email protected]. † Dendritic NanoTechnologies, Inc. ‡ Central Michigan University. 10.1021/nl034957m CCC: $27.50 Published on Web 04/13/2004

© 2004 American Chemical Society

by mass spectrometry,17-20 gel electrophoresis,21,22 and other analytical methods.21-23 Access to this level of macromolecular structure control has created substantial interest in the use of dendrimer structures as unimolecular mimics of globular proteins7,24,25 micelles,26-29 and a variety of other biological self-assemblies.4,30 Using strictly abiotic methods, it has been widely demonstrated over the past decade that dendrimers2 can be routinely constructed with control that rivals the structural regulation found in biological systems. Such mimicry and comparison of spherical dendrimers to proteins was made as early as 1990.4 The close scaling of size,7 shape,25,29 and quasi-equivalency comparison of dendrimer surfaces31-33 to nanoscale biostructures is both striking and provocative (Figure 1). These remarkable similarities suggest a broad strategy based on rational biomimicry for creating a repertoire of structure-controlled, size and shape-variable dendrimer assemblies.25,29 Successful demonstration of critical covalent chemistry to practice such a biomimetic approach has provided a versatile and powerful synthetic strategy for systematically accessing virtually any desired combination of size, shape, or surface chemistry in the nanoscale region. This is possible by combinatorial variation of critical dendritic module parameters such as (i) interior compositions, (ii) surfaces, (iii) generational levels, or (iv) architectural shapes (i.e., cone-like, spheroidal, ellipsoidal, rod-like, etc.). Substantial progress in this area has been reported by us recently.25,29 This work focused on “divergent synthesis

Figure 1. Comparison of micron-scale biological cells to nanoscale proteins and poly(amidoamine) dendrimers.

strategies” to produce “disulfide core, PAMAM dendrimers”. Specifically, we described reduction of the disulfide function, found in cystamine core dendrimers, to produce “single-site, thiol functionalized” (PAMAM) di-dendrons. Combinatorial hybridization of these single-site, sulfhydryl dendron components and reoxidation, provided a very versatile strategy for systematic shape designing and chemodifferentiation of the resulting dendrimer surfaces. The exquisite control of size, shape, and surface chemistry that is possible with dendrimers has already led to their use as replacements for proteins in a variety of applications. These uses include diagnostics,8 gene delivery,34 molecular weight calibrators,23,35 presentation of antigens,36 enzyme mimics,37 site isolation,24 globular protein mimics,29,35 etc.38 This letter reports a significant extension to this broad strategy and now involves self-assembly of DNA modified dendritic components. As described in the recent patent literature,39 single-site, single strand (ss DNA) functionalized di-dendrons can be routinely synthesized by conjugating various DNA sequences to single-site, thiol functionalized, di-dendrons.39 These structures represent a new class of linear-dendritic, architectural copolymers. We have now found that by selecting appropriate complementary DNA sequences for the linear portion of the architectural copolymer, these structures may be used as supramacromolecular40 components to produce novel, double stranded (dsDNA), core-shell nanostructures, possessing base-paired DNA cores surrounded by four dendrons as shown in Scheme 1. Furthermore, it has also been shown that a broad combinatorial, structure design strategy for the assembly of the dendritc components is possible, much as reported by Alivisatos et al.41 and others42-44 for analogous (ssDNA)functionalized quantum dots. As early as 1996, Alivisatos et al.45 and Mirkin et al.46 described the first self-assembly 772

of nanoparticles functionalized with complementary DNA sequences. Since that time innumerable reports have appeared describing the use of that strategy for the construction of two and three-dimensional nanoarchitectures by hybridization of single-site DNA functionalized nanocrystals.47 These selfassembled nanostructures have exhibited novel catalytic, optical and electronic properties.42 In some instances these constructs are being exploited in the development of new diagnostic and biodetection schemes.48,49 Synthesis of Single-Site, (ssDNA)-Functionalized Poly(amidoamine) Dendrimers. The single-site functionalized, dendrimer-(ssDNA) conjugates were synthesized as described earlier.39 The single-site sulfhydryl dendrons were prepared29 by reducing 10-7 moles of cystamine core PAMAM dendrimers (i.e., generation ) 2 or 3) possessing succinamic acid surfaces with 0.9 × 10-7 moles of dithiothreitol (DTT)(Acros) for >3 h in 200 µL of 1X PBS (150 M NaCl, 5.2 mM Na2HPO4, 1.7 mM NaH2PO4), pH ) 6.8, at room temperature. Depletion of DTT accompanied by reduction of the disulfide core to reactive, single-site, mercapto dendrons was verified using TLC and Ellman’s reagent to detect the presence of sulfhydryl groups. Reduced dendrons were used immediately for oligonucleotide coupling, before oxidation occurred to reform the cystamine core dendrimers. The targeted 32-base oligonucleotides to be coupled to the dendrons were synthesized based on a number of considerations. The sequences were introduced to provide a relatively strong base pair interaction, exhibiting a melting temperature of about 68 °C, while attempting to reduce the probability for self-interaction to form homodimers. Furthermore, the oligonucleotides were engineered with an EcoRI restriction endonuclease site at the midpoint of the sequence to allow for further analysis after attaching the Nano Lett., Vol. 4, No. 5, 2004

Scheme 1. (A) Scheme Describing (a)/(a′) Reduction of Disulfide, Thiol-functionalized dendri-(PAMAM) Di-dendrons, (b)/(b′) Bioconjugation of (ssDNA) (forward/reverse) to Produce Respective, Single-site (ssDNA)-(PAMAM) Di-dendrons and (c) Self-assembly of Respective (ssDNA)-(PAMAM) Di-dendron Conjugates by Watson-Crick Base Pairing to Produce (dsDNA) Core- (tetra-dendron) PAMAM Dendrimers with Differentiated Sizes (i.e., generations: Gx or Gy) and Surface Groups (X or Y). (B) Scheme Showing b/b′ Reaction for Conjugation of Oligonucleotides to Reduced Dendrons Using Sulfo-SMCC Crosslinker

dendron components. Lastly, they were designed to be complementary over only the thirty 3′ bases with two unpaired bases on their 5′ end. These two unpaired bases were introduced to function as a benign spacer between the dendron/ oligonucleotide junction in order to reduce steric interference by the dendron components. The two engineered (forward and reverse) sequences were obtained from Qiagen with a 5′ amine modification to facilitate coupling to the sulhydryl group of the reduced cystamine core dendrimer. Furthermore, the oligonucleotides were activated for coupling to dendrons by attaching a heterobifunctional cross-linker such as sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) (Pierce) to the 5′ amine modification. This was performed by incubating 10-8 moles of oligonucleotide with 10-6 moles of (sulfo-SMCC) in 400 µL 1X PBS, pH ) 7.2, for 1 h in the dark at room temperature. The pH of all reactions involving sulfo-SMCC must be controlled. At a pH > 7.5, the amine group can react with the maleimide group, giving rise to oligonucleotide dimers linked at their 5′ ends. Excess sulfo-SMCC was removed from the reaction by filtration through a 5000 MW cutoff regenerated cellulose filter (Amicon). The activated oligonucleotide was dissolved in 200 µL of 1X PBS, pH ) 6.8, to remove it from the filter. In view of the sulfo-SMCC cross-linker instability in aqueous Nano Lett., Vol. 4, No. 5, 2004

buffers, the activated oligonucleotide was coupled immediately to the reduced dendrons. Conjugation of oligonucleotides and dendrons was performed by mixing single-site, mercapto functionalized dendri-poly(amidoamine); generation ) 2.0 dendrons with the (forward) oligonucleotide and generation ) 3.0 dendrons with the (reverse) oligonucleotide. A 200 µL sample of the activated oligonucleotide was mixed with 200 µL of reduced dendrons and allowed to react overnight (16 h) at room temperature in the dark. The reaction was then terminated by centrifugal filtration through a 10 000 molecular weight cutoff membrane (Amicon) to remove excess dendron reagent followed by resuspension of the filter retentate in 50 µL of water. Since the 5′ amine modification of the oligonucleotides is not necessarily quantitative, uncoupled oligonucleotides were separated from the dendron-oligonucleotide conjugates by electrophoresis on a 4% TAE/agarose gel. The gel was stained with 0.02% methylene blue to visualize nucleic acids, after which the dendron-oligonucleotide conjugate was then excised from the gel and purified using a Mermaid-Spin kit (Q-Bio Gene). The recovered volume was dried overnight on a lyopholyzer and redissolved in 40 µL of water. Watson-Crick Base Pairing of (Single-Site, Functionalized (ssDNA)-dendri-PAMAM) Structures 4 and 5 to Form 6. Base-paired dendrimers were formed by combining and incubating appropriate dendrons coupled to their respective complementary oligonucleotides. A 5 µL sample of generation ) 3-(reverse) oligonucleotide and 5 µL of generation ) 2-(forward) oligonucleotide were heated at 95 °C and annealed by slowly cooling to room temperature over 2 h. Control reactions and electrophoretic analyses were performed with each of the oligonucleotide structures 1 and 2, the oligonucleotides base-paired together 3, and finally the Watson-Crick base paired dendron-oligonucleotide conjugates 6. A comparison of relative gel mobilities demonstrated the specificity of the base-paired dendrimer construct. Formation of the base-paired dendrimer was visualized by staining with the DNA specific acridine orange dye, after electrophoresis on a 10% polyacrylamide gel (Figure 3). The ability to base pair and the expected specificity of the (forward) and (reverse) oligonucleotides is evident in the left three lanes (i.e., lanes 1-3). Neither (forward) (lane 1) nor (reverse) (lane 2) single-strand oligonucleotide show a shift in gel mobility when electrophoresed independently. Base pairing occurs when (forward) and (reverse) oligonucleotides are mixed and annealed (lane 3), as shown by the slower migration of this construct. Likewise, the right three lanes (i.e., lanes 4-6) show the specific interaction of the G ) 2-(forward) conjugate with the G ) 3-(reverse) conjugate. Both G ) 2-(forward) (lane 4) and G ) 3-(reverse) (lane 5) conjugates exhibited the expected gel mobility. The lower, faint band in the G ) 2-(forward) construct is due to the presence of a small amount of uncoupled (forward) oligonucleotide. This comparison of relative structure mobilities by electrophoresis not only confirms the base pairing specificity of the oligonucleotides but also demonstrates there is no interaction between 773

Figure 2. Schematic of base pairing reaction. Two 5′ amine-modified oligonucleotides, forward (1) and reverse (2), are complimentary over 30 bases (blue) and contain a two base noncomplimentary spacer (red). These oligonucleotides were used in to make the dendrimeroligonucleotide conjugates G ) 2-forward (4) and G ) 3-reverse (5). Heating to 95 °C and slowly cooling to anneal allows a mix of oliognucleotides (1, 2) to anneal to specifically make double stranded DNA (3) and a mix of the dendrimer-oligonucleotide conjugates (4 and 5) to specifically make a base paired differentiated dendrimer (6).

Figure 3. Polyacrylamine gel electrophoresis (PAGE) analysis of base-paired dendrimers. Base pairing reactions were run on 10% PAGE to compare the relative mobilities of the forward and reverse oligonucleotides (lanes 1 and 2), the forward/reverse base-paired oligonucleotides (lane 3), the G ) 2-forward and G ) 3-reverse dendrimer-oligonucleotide conjugates (lanes 4 and 5), and the base-paired G ) 2-forward/G ) 3-reverse construct (lane 6).

the oligonucleotide and the anionic succinamic acid groups present on the surface of the conjugated dendrons. Had there been an interaction between the oligonucleotide and the dendron, at least one slower migrating species would have been observed on the gel. Upon annealing, there is a decrease in mobility of the major band, demonstrating self-assembly 774

of the G ) 2-(forward)/G ) 3-(reverse) base-paired, differentiated dendrimer (lane 6). The lower band in lane 6 corresponds in size to the G ) 2-(forward) construct that was in excess relative to the G ) 3-(reverse) conjugate. Since both conjugate constructs are derived from anionic, succinamic acid surface dendrimers and no higher molecular Nano Lett., Vol. 4, No. 5, 2004

Figure 4. MALDI-TOF analysis of single-strand DNA and DNA-dendron conjugates. MALDI-TOF mass spectrometry was performed using 3-hydroxypicolinic acid matrix to show the mass of forward (A) and reverse (B) single-strand oligonucleotides, G ) 3-reverse singlestrand dendron conjugate (C), G ) 2-forward single-strand dendron conjugate (D).

weight products are observed in the single construct reactions (lanes 4 and 5), it may be concluded that the G ) 2-(forward)/G ) 3-(reverse) Watson-Crick base pair is formed rather than a dendrimer/dendrimer or dendrimer/DNA assembly product. The double stranded construct G ) 2-(forward)/G ) 3-(reverse) was stable through several freeze thaws, at room temperature, and through an EcoRI restriction enzyme digest (37 °C, 3 h)(data not shown), indicating that the presence of the dendrons had little effect on the stability of the DNA duplex under the conditions used. MALDI-MS Analysis of Single-Site, (ssDNA)-Functionalized Poly(amidoamine) Dendrimers. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed to corroborate the PAGE evidence for the construction of the oligonucleotide-dendron conjugates. MALDI-MS is routinely performed on single-stranded DNA using 3-hydroxy picolinic acid (3-HPA) as a matrix.53 Positive ion MALDI-TOF mass spectra were recorded on a Bruker Daltonics Autoflex spectrometer in either the linear or reflectron mode using pulsed ion extraction. The acceleration voltage was 19 kV. The matrix solution (10 g/L 3-HPA in 30% acetonitrile) was mixed with the analyte solution (5 Nano Lett., Vol. 4, No. 5, 2004

uL matrix per 1 uL analyte), the mixture spotted on the target plate, and the spots allowed to dry at room temperature. The forward and reverse oligonucleotides show major peaks at the expected sizes with minor peaks at half the size (the doubly charged form) and at twice the size (nonspecific dimer)(Figure 4A and B). The peaks for the dendronoligonucleotide conjugates also appear at the predicted size (Figure 4C and D). The conjugate peaks are weaker and broader than those of oligonucleotide alone. This is evidently due to poor ionization and flight of the dendron in 3-HPA matrix as the G ) 3 conjugate gives consistently weaker and broader signals than the G ) 2 conjugate, indicating increased interference. In addition to the predicted peaks for the dendron-oligonucleotide conjugates, a peak correlating to the free oligonucleotide is also evident in these samples. These oligonucleotide peaks indicate either that the laser energy of the MALDI-MS cleaves some of the dendronoligonucleotide or that the low level of oligonucleotide contaminant in the samples ionizes and is detected better than the conjugates. Noncovalent structures, such as double-stranded DNA (dsDNA), are notoriously difficult to detect using MALDI775

MS, usually showing signals for only their component parts. Watson-Crick base-paired structures have been reported to be detected using the matrix 6-aza-2-thiothymine (ATT) with long oligonucleotides (70 and 80 bases)53,54 or using 3-HPA and preparing samples near 0 °C to decrease protonation of bases that denatures dsDNA.55 Several matrices that generate MALDI-TOF signals for either DNA or dendrimers include ATT, 3-HPA, 2,5-dihydroxybenzoic acid (DHB), 2′,4′,6′trihydroxyacetophenone (THAP), and 2-(4-hydroxyphenylazo)-benzoic acid (HABA). However, all of these matricies produced either no detectable signal or signal for only the single-standed components (data not shown). The inability to observe the dsDNA structure with only 30 complementary bases is not surprising given the observation of Kirpeckar et al. that there is a correlation between length of the double stranded region and the degree of dsDNA seen using ATT as a matrix.53 One of the ultimate challenges of “bottom-up nanosynthesis” is to be able to self-assemble appropriate components into unambiguous size, shape, and regiochemical specific nanostructures. Choi et al. have shown that oligonucleotides conjugated to the surface of complete dendrimers can be used to conjugate dendrimers of different generations.57 However, their method, with multiple oligonucleotides on each dendrimer surface, produces a statistical array of base-paired structures from which the desired product must be found. The method shown here couples a single oligonucleotide to a specific single site of a dendron. This results in specific structures being formed upon base pairing. Additionally, it also allows discrete chemistry to be performed with distinct dendron surfaces to permit nanoconstuction. Although this present work illustrates a simple binary example, namely, combination of size and regiochemical differenetiated dendritic components, it is the first step in demonstrating the endless combinatorial options that are possible with this approach.50-52 Acknowledgment. We acknowledge Dr. Douglas Swanson and Dr. David Hedstrand for their many helpful discussions and insights regarding this work, as well as Ms. Linda Nixon for figure and manuscript preparation, and the Army Research Laboratory (ARL) for partially funding this project (DAAD19-03-2-0012). References (1) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. (Tokyo) 1985, 17, 117132. (2) Fre´chet, J. M. J.; Tomalia, D. A. Dendrimers and Other Dendritic Polymers; John Wiley & Sons, Ltd.: West Sussex, 2001. (3) Tomalia, D.; Frechet, J. M. J. J. Polym. Sci. Part A: Polym. Chem. 2002, 40, 2719-2728. (4) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III Angew. Chem., Int. Ed. Engl. 1990, 29, 138-175. (5) Tomalia, D. A.; Mardel, K.; Henderson, S. A.; Holan, G.; Esfand, R. In Handbook of Nanoscience, Engineering and Technology; Goddard, W. A., III, Brenner, D. W., Lyshevski, S. E., Iafrate, G. J., Eds.; CRC Press: Boca Raton, 2003, p 20.21-20.34. (6) Tomalia, D. A. AdV. Mater. 1994, 6, 529-539. (7) Esfand, R.; Tomalia, D. A. Drug DiscoVery Today 2001, 6(8), 427436. (8) Singh, P. In Dendrimers and Dendritic Polymers; Fre´chet, J. M. J., Tomalia, D. A., Eds.; J. Wiley & Sons, Ltd.: West Sussex, 2001; pp 463-484. 776

(9) Guo, W.; Li, J. J.; Wang, A. W.; Peng, X. J. Am. Chem. Soc. 2003, 125, 3901-3909. (10) Gopidas, K. R.; Whitesell, J. K.; Fox, M. A. J. Am. Chem. Soc. 2003, 125, 6491-6502. (11) Newkome, G. R.; Moorfield, C. N.; Vo¨gtle, F. Dendritic Molecules; VCH: Weinheim, 1996. (12) Zeng, F.; Zimmerman, S. C. Chem. ReV. 1997, 97, 1681-1712. (13) Tomalia, D. A. Sci. Am. 1995, 272, 62-66. (14) Fre´chet, J. M. J. Science 1994, 263, 1710-1715. (15) Matthews, O. A.; Shipway, A. N.; Stoddart, J. F. Prog. Polym. Sci. 1998, 23, 1-56. (16) Lothian-Tomalia, M. K.; Hedstrand, D. M.; Tomalia, D. A. Tetrahedron 1997, 53, 15495-15513. (17) Kallos, G. J.; Tomalia, D. A.; Hedstrand, D. M.; Lewis, S.; Zhou, J. Rapid Commun. Mass Spectrom. 1991, 5, 383-386. (18) Dvornic, P. R.; Tomalia, D. A. Macromol. Symp. 1995, 98, 403428. (19) Tomalia, D. A.; Durst, H. D. In Supramolecular Chemistry I Directed Synthesis and Molecular Recognition; Weber, E. W., Ed.; Springer-Verlag: Berlin, 1993; pp 193-313. (20) Hummelen, J. C.; van Dongen, J. L. J.; Meijer, E. W. Chem. Eur. J. 1997, 3, 1489-1493. (21) Brothers, H. M., II; Piehler, L. T.; Tomalia, D. A. J. Chromatogr. A 1998, 814, 233-246. (22) Zhang, C.; Tomalia, D. A. In Dendrimers and Other Dendritic Polymers; Fre´chet, J. M. J., Tomalia, D. A., Eds.; John Wiley & Sons: West Sussex, 2001; pp 239-252. (23) Dubin, P. L.; Edwards, S. L.; Mehta, M. S.; Tomalia, D. J. Chromatogr. 1993, 635, 51-60. (24) Hecht, S.; Fre´chet, J. M. J. Angew. Chem., Int. Ed. 2001, 40(1), 7491. (25) Tomalia, D. A.; Brothers, H. M., II; Piehler, L. T.; Durst, H. D.; Swanson, D. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99(8), 50815087. (26) Tomalia, D. A. Macromol. Symp. 1996, 101, 243-255. (27) Turro, N. J.; Chen, W.; Ottaviani, M. F. In Dendrimers and Other Dendritic Polymers; Fre´chet, J. M. J., Tomalia, D. A., Eds.; John Wiley & Sons: West Sussex, 2001; pp 309-330. (28) Watkins, D. M.; Sayed-Sweeet, Y.; Klimash, J. W.; Turro, N. J.; Tomalia, D. A. Langmuir 1997, 13, 3136-3141. (29) Tomalia, D. A.; Huang, B.; Swanson, D. R.; Brothers, H. M., II; Klimash, J. W. Tetrahedron 2003, 59, 3799-3813. (30) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897-4902. (31) Percec, V.; Johansson, G.; Ungar, G.; Zhou, J. P. J. Am. Chem. Soc. 1996, 118, 9855-9866. (32) Percec, V.; Ahn, C.-H.; Unger, G.; Yeardly, D. J. P.; Moller, M. Nature 1998, 391, 161-164. (33) Hudson, S. D.; Jung, H.-T.; Percec, V.; Cho, W.-D.; Johansson, G.; Ungar, G.; Balagurusamy, V. S. K. Science 1997, 278, 449-452. (34) Eichman, J. D.; Bielinska, A. U.; Kukowska-Latallo, J. F.; Donovan, B. W.; Baker, J. R., Jr. In Dendrimers and Other Dendritic Polymers; Fre´chet, J. M. J., Tomalia, D. A., Eds.; John Wiley & Sons Ltd.: West Sussex, 2001; pp 441-461. (35) Chow, H.-F.; Mong, T. K.-K.; Chang, Y.-H.; Cheng, H. K. Tetrahedron 2003, 59, 3815-3820. (36) Tam, J. P.; Lu, Y.-A. Proc. Natl. Acad. Sci. U.S.A. 1989, 85, 90849088. (37) Liu, L.; Breslow, R. J. Am. Chem. Soc. 2003, 125, 12110-12111. (38) Kasai, S.; Nagasawa, H.; Shimamura, M.; Uto, Y.; Hori, H. Bioorg. Medicinal Chem. Lett. 2002, 12, 951-954. (39) Klimash, J. W.; Brothers, H. M., II; Swanson, D. R.; Yin, R.; Spindler, R.; Tomalia, D. A.; Hsu, Y.; Cheng, R. C.; U.S. Patent 6,020,457, 2000. (40) Tomalia, D. A.; Majoros, I. In Supramolecular Polymers; Ciferri, A., Ed.; Marcel Deker: New York, 2000; Chapter 9, pp 359-434. (41) Loweth, C. J.; Caldwell, W. B.; Peng, X.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1999, 38, 1808-1812. (42) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (43) Mbinkyo, J. K. N.; Reiss, B. D.; Martin, B. R.; Keating, C. D.; Natan, M. J.; Mallouk, T. E. AdV. Mater. 2001, 1, 249-254. (44) Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem., Int. Ed. 2001, 40, 1861-1864. (45) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, J. M. P. Nature 1996, 382, 609-611. Nano Lett., Vol. 4, No. 5, 2004

(46) Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558-1562. (47) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862. (48) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (49) Wei, Y.; Cao, C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 15361540. (50) Cao, Y. W.; Jin, R.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 7961-7962. (51) Shchepinov, M. S.; Udalov, I. A.; Bridgman, A. J.; Southern, E. M. Nucleic Acids Res. 1997, 25, 4447-4454. (52) Shchepinov, M. S.; Mir, K. U.; Elder, J. K.; Frank-Kamenetski, M. D.; Southern, E. M. Nucleic Acids Res. 1999, 27, 3035-3041.

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(53) Kirpeckar, F.; Berkenkamp, S.; Hillenkamp, F. Anal. Chem. 1999, 71, 2334-9. (54) Lecchi, P.; Pannell, L. K. J. Am. Soc. Mass Spectrom. 1995, 6, 972-5. (55) Nordhoff, E.; Cramer, R.; Kara, M.; Hillenkamp, F.; Kirpeckar, F.; Kristiansen, K.; Roepstorff, P. Nucleic Acids Res. 1993, 21, 334757. (56) Little, D. P.; Jacob, A.; Becker, T.; Braun, A.; Darnhofer-Demar, B.; Jurinke, C.; van den Boom, D.; Ko¨ster, H. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 323-30. (57) Choi, Y.; Mecke, A.; Orr, B. G.; Banaszack Holl, M. M.; Baker, J. R., Jr. Nano Lett. 2004, 4, 391-397.

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