Conformation of Oligonucleotides Attached to Gold Nanocrystals

nanocrystals with different DNA/Au ratios (1, 2, ..., saturation). The electrophoretic mobility of these conjugates was determined on 2% agarose gels,...
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Conformation of Oligonucleotides Attached to Gold Nanocrystals Probed by Gel Electrophoresis

2003 Vol. 3, No. 1 33-36

Wolfgang J. Parak,*,† Teresa Pellegrino, Christine M. Micheel, Daniele Gerion, Shara C. Williams, and A. Paul Alivisatos* Materials Science DiVision, Lawrence Berkeley National Laboratory, and Department of Chemistry, UniVersity of California, Berkeley, California Received November 11, 2002

ABSTRACT Thiol-modified single stranded oligonucleotides of different lengths (8 to 135 bases) were attached to the surface of 10 nm diameter Au nanocrystals with different DNA/Au ratios (1, 2, ..., saturation). The electrophoretic mobility of these conjugates was determined on 2% agarose gels, and the effective diameter of the DNA/Au conjugates was determined. This diameter depends on the conformation of the oligonucleotides adsorbed on the Au surface. For low surface coverage, nonspecific wrapping of the DNA around the nanoparticles was observed. For high surface coverage, short oligonucleotides were shown to be oriented perpendicular to the surface and fully stretched. For high surface coverage and long oligonucleotides, the inner part close to the Au surface was determined to be fully stretched and pointed perpendicular to the surface, whereas the outer part adopts random coil shape.

Conjugates of nanocrystals with oligonucleotides are of current interest both due to the potential for using the programmability of DNA base-pairing to spatially organize nanocrystals, and due to the multiple mechanisms by which the nanocrystals provide a strong signature for detection of particular DNA sequences.1-3 Generally, the ends of single stranded oligonucleotides are attached to the nanocrystals, in the case of Au directly via thiol-linkage,4 and then the nanocrystal-oligonucleotide conjugate is assumed to be available to hybridize to the complimentary strand. It is of considerable interest to know more about the nature of the conformation of Au nanocrystals conjugated to oligonucleotides. Intuitively three types of interaction have to be considered. Nonspecific interaction between the individual nucleotides and the gold surface favors wrapping of the DNA around the nanocrystals. The energy gained by forming a thiol-gold bond instead favors the covalent attachment of as many DNAs as possible with their thiolated ends to the gold surface, and with the rest of the oligonucleotides directed toward the surrounding solution. Entropy favors the dangling part of the oligonucleotides to adopt a random coil confirmation. Gain in binding energy for thiol-gold bond formation on the other hand favors a stretched configuration of the dangling part of the oligonucleotide, so that DNA can be packed onto a nanocrystal close to the geometric limit. * Corresponding authors: e-mail [email protected], [email protected]. † Present address: Center for Nanoscience, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany. 10.1021/nl025888z CCC: $25.00 Published on Web 12/03/2002

© 2003 American Chemical Society

Figure 1. Different possible configurations of DNA molecules attached to the surface of Au nanocrystals.

Therefore we can imagine three limiting cases: DNA can be wrapped around the nanocrystal, bound in random coiled shape, or bound in stretched shape pointing perpendicular to the surface, see Figure 1. The conformation and packing of the DNA can strongly influence the accessibility of the oligonucleotides for hybridization.5 To address these issues we have performed a comprehensive study of the electrophoretic mobility of Au nanocrystal/DNA conjugates as a function of the number of oligonucleotides bound to the nanocrystal (from 1 to saturation) and as a function of the length of the oligonucleotides (from 8 to 135 bases). Here we present results only for 10 nm nanocrystals, but a more comprehensive study as a function of nanocrystal size is in progress. Au nanocrystals have been chosen for this study for two reasons. First, the use of Au/DNA conjugates for oligonucleotide detection has been established both for nanoparticles

with discrete numbers of DNA attached6 and for nanocrystals with saturation coverage of DNA.7 Second, by coating Au nanocrystals with negatively charged triphenyl phosphine sulfonate8 their charge density becomes similar to that of DNA. As a consequence, when oligonucleotides are attached to the Au, the primary effect is to change the size of the conjugate, but not its charge density, so that the electrophoretic mobility is a rather direct surrogate for size.4 We note that this is not the case for all types of nanoparticles. For instance, for silica coated nanocrystals with low charge density compared to DNA, the mobility is increased upon addition of DNA.9 Finally, we have employed electrophoresis as a probe because it is extremely sensitive to conformation and because the same experimental technique can be applied across the very wide range of samples studied here. We note in this context that while fluorescence quenching is a powerful probe for oligonucleotide conformations, the use here is exceedingly complex. This is because the Au nanoparticle may enhance or quench the dye fluorescence, depending upon the size and the precise distance of the dye from the Au particle. Further, dyes attached to densely packed oligonucleotide layers may exhibit energy transfer and J-aggregate phenomena.10 A geometric sorting technique such as electrophoresis can provide important information about the average conformation. The mobility of the Au is always retarded by the addition of oligonucleotides, see Figure 2. Figure 3 shows the mobility of the conjugates as a function of both the number of oligonucleotides attached (from 1 to saturation), and as a function of the length of the single-stranded DNA. For each length of DNA, the mobility is altered most by the first addition of a single strand. The progressive addition of more strands yields a ladder of mobilities that eventually converges to the saturation limit. For a fixed number of single-stranded DNAs attached, the mobility is progressively reduced as the DNA strands become longer. To convert these results into a measure of the size of the Au/DNA conjugates we compared the results presented in Figure 3 to the mobility of phosphine-coated Au nanocrystals of differing size, but with no oligonucleotides attached. The reliability of this method of size calibration is demonstrated by considering the behavior of the Au conjugates with saturation coverage of single-stranded DNA as a function of the length of the oligonucleotides, see Figure 4. When the strands are short, the Au/DNA conjugates are equal to the sum of the Au nanocrystal diameter plus twice the length of the fully stretched single-stranded DNA. This means that for short conjugates (less than around 30 bases) the DNA is fully stretched and densely packed. For saturation for conjugates longer than about 30 bases, the size of the conjugate is progressively smaller than the maximum possible size of the Au diameter plus twice the stretched DNA length. The Au/DNA conjugate diameter for these long oligonucleotides can be well described by assuming a configuration of the DNA in which the first 30 bases of the oligonucleotide are fully stretched and a random coil shape of its subsequent part, see Figure 4. This suggests that for saturation coverage of the conjugates, there is a critical length 34

Figure 2. 2% agarose gel loaded with different DNA/Au conjugates after 90 min @ 100 V in 0.5× TBE buffer (ca. 50 mM). Prior to running the gel, 54-base DNA with a 5′ thiol modification was added in different ratios (cadded,max(DNA) ) 690 mM) to phosphine coated Au nanocrystals of 10 nm diameter (c(Au) ) 0.69 mM) and incubated for 1 h in 25 mM phosphate buffer (pH ) 7.0) with 50 mM NaCl on a rocking platform at room temperature. To obtain the different DNA/Au ratios, the DNA concentration was reduced successively by a factor of 2 by dilution with H2O, before the DNA was added to the Au nanocrystals. Because of progressive dilution errors, the DNA/Au ratio given on the left side of the gel has to be considered as estimate, especially for small DNA/Au ratios. Furthermore, the DNA/Au ratio given on the left side of the gel is the number of DNA molecules that have been added into solution per Au nanoparticle. Since not all DNA molecules can bind, especially not at very high concentration when the surface of the Au nanocrystals is already completely covered with DNA, this number is higher than the number of DNA molecules that are actually bound per Au nanocrystal. Depending on the DNA/Au ratio, bands with a discrete number of DNA molecules (1, 2, 3, ...) attached per nanocrystal can be resolved. After a certain amount of DNA is added, the nanocrystals are saturated (sat) with DNA and there is no further retardation in mobility upon further addition of DNA. At one point the surface of the particles is saturated and no more DNA molecules can bind. The relative mobility is determined as mobility of any conjugate referring to the mobility of free Au nanocrystals (relative mobility of free Au nanocrystals ) 1, retarded mobility for DNA/Au conjugates < 1).

beyond which the outermost portion of the DNA will be much more accessible to hybridization. Studies of discrete conjugates with only one or a few oligonucleotides attached show interesting effects as well, see Figure 4. For longer oligonucleotides, the conjugate diameters are smaller than expected from the sum of a globular single-stranded DNA plus the Au nanocrystal. This is consistent with a picture in which the oligonucleotide is wrapped around the particle. This reflects a strong interaction between the nanocrystals and the DNA, again influencing the hybridization efficiency. The group of Mirkin and Letsinger recently reported that the affinity of single nucleotides to Au nanocrystals is biggest for cytosine (C) and guanine (G), smaller for adenine (A), and smallest for thymine (T).11 Our results strongly support this. We found in our gel-electrophoresis experiments for oligonucleotides with 36 bases that raising the content of A and T from 36% to 64% and the content for T alone from 19% to 25% Nano Lett., Vol. 3, No. 1, 2003

Figure 3. Relative mobility of DNA/Au conjugates, depending on the length of the DNA and the number of single-stranded oligonucleotides attached per particle (0, 1, 2, ..., sat).

decreased the relative saturation mobility of the conjugates from 0.54 to 0.49, which corresponds to an increase in diameter. For oligonucleotides with 43 bases, incrementing the content of A and T from 35% to 53% and the content of T alone from 21% to 28% reduced the relative mobility of the DNA-saturated Au nanocrystals from 0.55 to 0.41, corresponding again to increased conjugate diameters. Furthermore, bands with discrete numbers of oligonucleotides attached per nanocrystals could be resolved for the 43-base DNA with 28% T, but not for the one with 21% T. These results suggest that for oligonucleotides with low content of A and T there is a significant tendency to wrap around the Au nanocrystals, because of the interaction between Au and the C and G. For only a few DNAs per nanocrystal, this ill-defined nonspecific binding causes smeared out bands and thus complicates the resolution of conjugates with a discrete number of oligonucleotides bound per particle on a gel. In the case of conjugates saturated with DNA, partial wrapping of the DNA around the particles reduces the overall diameter of the conjugates. Oligonucleotides rich in A and T, on the other hand, show a reduced tendency to nonspecifically wrap around Au nanocrystals. In the limit of saturation, the part of the DNA close to the thiol-Au bond is fully stretched and points perpendicular to the Au surface, which allows for the maximum possible surface coverage. This results in an increment of the conjugate diameter. We conclude that for 10 nm diameter Au nanocrystals saturated with DNA, the inner part of about 30 bases of the oligonucleotides is fully stretched, whereas the outer parts adopt random coil configuration. In the case of only a few DNAs per nanocrystal, fragments of the oligonucleotides are nonspecifically attached to the Au surface. These results have implications for effective hybridization to the conjugates. Au nanocrystals fully covered with oligonucleotides of at least 45 bases length should be effective building blocks, especially when using only the code of the outer 15 bases for hybridization. Wrapping of the DNA around the particle surface is omitted and the tight DNA coating ensures Nano Lett., Vol. 3, No. 1, 2003

Figure 4. Effective diameter of DNA/Au conjugates, derived from the relative mobilities shown in Figure 3 and a calibration curve (data not shown) of the mobility of unconjugated Au nanocrystals of different size, which relates mobilities to sizes. Three theoretical curves are plotted to fit the diameter of conjugates saturated with DNA. The determined diameter is always assumed to be 10 nm diameter of the Au nanocrystal and 2 times the extension of the DNA molecules. The extension of the DNA molecules comprises 0.92 nm accounting for the thiol-group at the 5′-end, which is linked by a carbohydrate spacer of 6 carbon atoms (5′ C6-spacer: S-trityl6-mercaptohexyl, cfg. http://www.idtdna.com). In the case of stretched DNA the extension of the DNA is assumed to be its contour length. A value of 0.43 nm per base for single-stranded was used to calculate the respective contour lengths.13 In the case of coiled DNA the extension is assumed to be 2 times the radius of gyration. In first approximation the radius of gyration is the square root of one-third of the product of contour length and persistence length.13 For single-stranded DNA in 50 mM buffer a persistence length of 2 nm was used.13 It has to be pointed out that experimental values for the persistence length of single-stranded DNA show significant variation depending on the used method (compare, e.g., the values found by Tinland et al.13 with those reported by Smith et al.14). In a combined model, the extension of the first 30 bases was taken as their contour length () stretched DNA), whereas the extension of all following bases was derived by 2 times their radius of gyration () coiled DNA).

excellent stability even for high salt concentrations. The outer bases are close to the random coil shape of free DNA molecules and should therefore be highly accessible for hybridization. To obtain building blocks with only one coding DNA per nanocrystal we suggest using nanocrystals coated with a dense layer of short oligonucleotides of about 30 bases length, and one single, long oligonucleotide of which only the outer part is used for hybridization, so that the shell of short oligonucleotides prevents wrapping of the coding parts of the DNA around the surface. This idea is similar to concepts used for oligonucleotides adsorbed on planar gold surfaces.12 Acknowledgment. W.J.P. was supported in part by German Research Foundation (DFG). T.P. was supported in part by the University of Bari, Italy. C.M.M. is a Howard Hughes Medical Institute Predoctoral Fellow. This work was supported (grants to A.P.A.) by the NIH National Center for Research Resources, Grant Number 1 R01 RR-1489135

01 through the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, DOD Advanced Research Projects Agency (DARPA) under Grant No. N00014-99-1-0728, Princeton, and [in part] by the Director, Office of Energy Research, Office of Science, Division of Materials Sciences, of the U.S. Department of Energy under Contract No. DEAC03-76SF00098. The views expressed in this paper are not endorsed by the sponsor. References (1) Mirkin, C. A. A DNA-Based Methodology for Preparing Nanocluster Circuits, Arrays, and Diagnostic Materials. MRS Bull. 2000, 25, 4354. (2) Niemeyer, C. M. Self-assembled nanostructures based on DNA: towards the development of nanobiotechnology. Curr. Opin. Chem. Biol. 2000, 4, 609-618. (3) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. Electrophoretic and Structural Studies of DNADirected Au Nanoparticle Groupings. J. Phys. Chem. B 2002, 106, 11758-11763. (4) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P. Electrophoretic isolation of discrete Au nanocrystal/DNA conjugates. Nano Lett. 2001, 1, 32-35. (5) Pen˜a, S. R. N.; Raina, S.; Goodrich, G. P.; Fedoroff, N. V.; Keating, C. D. Hybridization and Enzymatic Extension of Au NanoparticleBound Oligonucleotides. J. Am. Chem. Soc. 2002, 124, 7314-7323.

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(6) Maxwell, D. J.; Taylor, J. R.; Nie, S. Self-Assembled Nanoparticle Probes for Recognition and Detection of Biomolecules. J. Am. Chem. Soc. 2002, 124, 9606-9612. (7) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. The DNAMediated Formation of Supramolecular Mono- and Multilayered Nanoparticle Structures. J. Am. Chem. Soc. 2000, 122, 6305-6306. (8) Schmid, G.; Lehnert, A. The complexation of gold colloids. Angew. Chem., Int. Ed. Engl. 1989, 28, 780-781. (9) Parak, W. J. et al. Conjugation of DNA to silanized colloidal semiconductor nanocrystaline quantum dots. Chem. Mater. 2002, 14, 2113-2119. (10) Hranisavljevic, J.; Dimitrijevic, N. M.; Wurtz, G. A.; Wiederrecht, G. P. Photoinduced Charge Separation Reactions of J-Aggregates Coated on Silver Nanoparticles. J. Am. Chem. Soc. 2002, 124, 45364537. (11) Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Sequence-Dependent Stability of DNA-Modified Gold Nanoparticles. Langmuir 2002, 18, 6666-6670. (12) Herne, T. M.; Tarlov, M. J. Characterization of DNA Probes Immobilized on Gold Surfaces. J. Am. Chem. Soc. 1997, 119, 89168920. (13) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Persistence Length of Single-Stranded DNA. Macromolecules 1997, 30, 5763-5765. (14) Smith, S. B.; Cui, Y.; Bustamante, C. Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules. Science 1996, 271, 795-799.

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Nano Lett., Vol. 3, No. 1, 2003