J. Phys. Chem. C 2008, 112, 89-94
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Hybridization of ssDNA with a Complementary DNA Probe Tethered to a Gold NanoparticlesEffect of Steric Hindrance Caused by Conformation Yoshihiro Takeda,*,† Tamotsu Kondow,‡ and Fumitaka Mafune´ £ East Tokyo Laboratory, Genesis Research Institute, Inc., 717-86 Futamata, Ichikawa, Chiba 272-0001, Japan, Cluster Research Laboratory, Toyota Technological Institute, 717-86 Futamata, Ichikawa, Chiba 272-0001, Japan, and Department of Basic Science, Graduate School of Arts and Sciences, The UniVersity of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan ReceiVed: October 7, 2006; In Final Form: March 11, 2007
We investigated hybridization of probe DNA I and II tethered to gold nanoparticles (bulky probe I and II, respectively) with target single-strand DNA (ssDNA), where DNA I and II have the sequences complementary to the 50mer sequence starting from the 3501st (center) nucleotide and the 7199th (terminus) one of the target ssDNA (7249 bases), respectively. The hybridization rate of the bulky probe I was found to be slower than that of the bulky probe II. This finding is explained in terms of steric hindrance of the bulky probes. The steric hindrance is more significant when the probe hybridizes with a target ssDNA at a site nearer to its center. The freely jointed chain model is employed to analyze the hybridization kinetics semiquantitatively. The analysis shows that the ratio of the rate constant at the center to that at the terminus is magnified more when a bulkier probe is used.
1. Introduction Oligonucleotides tethered to solid supports such as nanoparticles have been utilized in recent years for a wide variety of applications including detection of genetic polymorphisms,1-4 disease screening5,6 and diagnosis,7 purification of biomolecules,8 monitoring gene expression,9-11 analysis of genome,12,13 and so forth. In these applications, hybridization of nucleic acid targets with tethered oligonucleotide probes is the central event. Many attempts have been made to identify important factors which control a hybridization efficiency of oligonucleotides tethered to solid supports.14-17 Among such factors, steric hindrance is considered to be one of the most important ones and becomes particularly crucial when the oligonucleotide probes are tethered too densely to the solid supports. The rate of hybridization of the probes to a target changes greatly with the probe density on the supports, and simultaneously, the probe density influences both the efficiency of duplex formation and the rate of hybridization because an increase of the probe density results in an increasing degree of steric hindrance.18-20 Let us consider hybridization of such a probe DNA to a target ssDNA. The hybridization is obviously retarded by steric hindrance due to the bulky three-dimensional conformation of the target ssDNA. In fact, the degree of the steric hindrance is greatly reduced when the target ssDNA is stretched to be onedimensional from three-dimensional in conformation, and as a result, the target ssDNA hybridizes more readily with the probe DNA.21,22 In addition, the probe itself sterically hinders the hybridization as well. In summary, the hybridization rate changes significantly by steric hindrance due to the bulkiness of the probe and the target. * To whom correspondence should be addressed. E-mail: takeda@ clusterlab.jp. † Genesis Research Institute. ‡ Toyota Technological Institute. £ The University of Tokyo.
In this paper, we report the hybridization kinetics of probe DNAs tethered to a gold nanoparticle (“bulky probe”) to a target ssDNA containing the complementary sequence to the prove DNA. The hybridization rate of bulky probes having the complementary sequence at the center of the target ssDNA was found to be smaller than that of bulky probes having the complementary sequence at the terminus. The hybridization kinetics was analyzed by using the freely jointed chain model. It was shown that (1) the steric hindrance of the bulky probe and the target ssDNA engenders retardation of the hybridization rate at the center, and (2) the hybridization rate at the terminus differs more significantly from that at the center by using a bulkier probe. 2. Experimental Section Chemicals and Oligodeoxyribonucleotides. A commercially available M13 mp18 single-strand DNA (Takara Bio, Inc.) and other chemicals employed in the present experiment were used without further purification. DNA I and II, 5′-(alkanethiol) oligonucloetides (50mer), were synthesized by a DNA synthesizer and purified by HPLC. The sequences of DNA I and II are complementary to the 50mer sequence starting from the 3501st and the 7199th nucleotide of the M13 mp18 single-strand DNA (ssDNA), respectively, as shown in Figure 1. Preparation of the Bulky Probe. Gold nanoparticles with a diameter of ∼13 nm were prepared by reduction of HAuCl4 by citrate.23 An aqueous solution of HAuCl4 (1 mM, 10 mL) was brought to reflux while stirring, and then, 1 mL of a 38.8 mM tricitrate solution was added quickly. The color of the solution changed from pale yellow to deep red. After the color changed, the solution was kept refluxed for an additional 15 min and then was cooled down to room temperature. The solution was filtered by a Micron Separation Inc. nylon filter with a pore of 0.45 µm. Absorption spectra of the solution containing gold nanoparticles with diameters of ∼13 nm were observed. The
10.1021/jp0665980 CCC: $40.75 © 2008 American Chemical Society Published on Web 12/13/2007
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Figure 1. Sequences of the 5′-(alkanethiol) oligonucloetide probes DNA I and II tethered to a gold nanoparticle and their complementary parts of a target M13 mp18 single-strand DNA used for the present experiment. The numbers surrounded by the squares indicate the nucleotide number from the 5′ terminus of the target ssDNA.
spectra exhibit a peak at the wavelength of 518-520 nm due to surface plasmon excitation. DNA I and DNA II tethered to the gold nanoparticles (“bulky probe I” and “bulky probe II”, respectively) were synthesized as follows: 5 mL of an aqueous gold nanoparticle solution (∼17 nM) was mixed with an aqueous solution containing DNA I (or II), where the initial DNA concentration was set to be such a value that the final DNA concentration became 2.5 µM. After 16 h, the solution was mixed with a buffer solution to reach the final buffer concentration of 0.1 M NaCl and 10 mM phosphate (pH 7) and was allowed to stand for 40 h in order to remove excess reagents from the solution thus prepared; the solution was centrifuged for 60 min at least at 15000 rpm. After removing the supernatant, the resulting red oily precipitate was washed three times with 1 mL of a buffer solution containing 0.1 M NaCl and 10 mM phosphate buffer (pH 7). Hybridization of Target ssDNA with the Bulky Probe. Target ssDNAs were hybridized with bulky probes by placing an aqueous solution containing the bulky probes (5.4 × 10-8 M) and the target ssDNA (1.8 × 10-8 M) in a tube at the temperature of 30 C°. The EtBr-precast agarose gel electrophoresis was used to determine the degree of hybridization by measuring the intensity of a dark-red band appearing in a 1% agarose gel, which results from light scattering of hybrids of the bulky probes with the target ssDNAs. Under illumination of UV light, the hybrids in the dark-red band were found to fluoresce when the hybrids in the band were intercalated with EtBr dyes. 3. Results Figure 2a shows a typical picture of a 1% agarose gel obtained by electrophoresis of an aqueous solution containing bulky probes, target ssDNAs, and hybrids of the bulky probes with the targets’ ssDNA after the solution was incubated at the temperature of 30 C° for different incubation times. Figure 2b shows a typical picture of the same agarose gel illuminated by
UV light. Lanes 1-7 in panels a and b correspond to electrophoresis of the solution containing bulky probes II, and lanes 8-14 correspond to that of the solution containing bulky probes I. The lane marked by “Ref” is given by a solution containing only the target ssDNAs. An incubation time is marked above each lane, as shown in panel a. The arrow indicates the position of the hybrids in each lane. The color of the band is dark red due to scattering of daylight by gold nanoparticles in the hybrids. As shown in panel b, the band also indicated by the arrow fluoresces under illumination of UV light from EtBr dyes intercalated with the hybrids. As the incubation time increases, the intensity of the band for the bulky probe II increases more rapidly than that for the bulky probe I (see panel a). In each lane, several bands located above the band of the hybrids are ascribed to gold nanoparticles bound with more than one target ssDNA per gold nanoparticle. Aggregation of the gold nanoparticles hardly occurs during the experiment because the surface of the gold nanoparticles is covered with probe DNAs so densely that the surface is negatively charged, and as a result, the gold nanoparticles are repelled from each other due to negative charges on them. Gold nanoparticles incompletely covered with probe DNAs are aggregated and hence are removed in the preparation stage by centrifugation. Let us define a degree of hybridization (Hyb(t)) as
Hyb(t) )
I(t) I0
(1)
where I(t) represents the intensity of a band due to hybrids at an incubation time, t (min), and I0 represents the intensity of the band at the incubation time of 1260 min, when target ssDNAs are considered to be completely hybridized. Figure 3 shows a degree of hybridization as a function of the incubation time, where the open squares and circles represent the degree of hybridization for bulky probes I and II, respectively. The free energies for formation of duplexes of the hybrids
Hybridization of DNA Tethered to Gold Nanoparticle
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Figure 3. The degree of hybridization plotted against the incubation time. The open squares and the open circles show the degrees of hybridization of bulky probes I and II, respectively. The solid lines represent the best-fit curve to the data obtained by using eq 3.
Figure 2. Pictures of 1% agarose electrophoresis gels (panel a) and the same agarose electrophoresis gels illuminated by UV light (panel b). The lane marked by “Ref” is for a solution containing only target ssDNAs. Incubation times for these solutions are marked above the lanes. The arrows in panels a and b indicate the positions of bulkytarget hybrids in the agarose electrophoresis gels. The probe-target hybrids form the dark-red band in panel a, which also emitted fluorescence as well from EtBr dyes intercalated in the hybrids in panel b.
made of the bulky probes I and II are calculated to be -57.5 and -53.8 kcal/mol, respectively, by using the algorithm established by Zuker.24 The dissociation constants Kd of the duplexes made of the bulky probes I and II are smaller than 10-38 at the temperature of 30 °C. Therefore, the hybridization reaction of a bulky probe (P) with a target ssDNA (T) is approximated by an irreversible second-order process as25 k
P + T 98 PT
(2)
where k represents the rate constant of the hybridization and PT represents a hybrid of P with T. The rate equation for the formation of PT is
d[PT] ) k[P][T] dt
(3)
where [X] represents the concentration of X in the solution. With [P]0 and [T]0 as the initial concentrations of P and T, respectively, the concentrations of [P] and [T] at a given time, t, are given by [P] ) ([P]0 - [PT]) and [T] ) ([T]0 - [PT]), respectively. Hence, the rate equation is solved as
Hyb(t) ) A
[P]0(exp(([T]0 - [P]0)kt) - 1) [T]0exp(([T]0 - [P]0)kt) - [P]0
(4)
where A is a constant. The solid curves in Figure 3 show those calculated from eq 4 with an adjustable parameter kb1 (hybridization rate constant for the bulky probe I) of (3.92 ( 0.53) × 102 mol-1‚s-1 and that of kb2 (hybridization rate constant for the bulky probe II) of (1.66 ( 0.26) × 104 mol-1‚s-1. The calculations reproduce the dependencies of the degree of hybridization on the incubation time. The value, [PT], was obtained from the band intensities of the hybrids including one target ssDNA per gold nanoparticle and those including more than one target ssDNA per gold nanoparticle. When a bulky probe I was used, the main hybrids were the gold nanoparticles bound to one target ssDNA during
the hybridization reaction. In the hybridization reaction including a bulky probe I, each bulky probe I hybridizes mainly with one target ssDNA. Therefore, the hybridization rate constant for the bulky probe I calculated from eq 4 should be almost equal to the hybridization rate constant calculated with consideration of formation of a bulky probe I bound to more than one target ssDNA. When a bulky probe II is used, the band of the bulky probe II bound to one target ssDNA appears at an early incubation time. On the other hand, the bands of gold nanoparticles bound to more than two target ssDNA are even weaker at the incubation time of 30 min. Therefore, [PT] can be approximated by the intensities of all of the bands, and eq 4 can be used to obtained the rate constant of the hybridization. 4. Discussion Model of Hybridization Kinetics. As shown in the previous section, the hybridization rate constant kb2 is 42.2 times as large as the rate constant kb1, mainly because a bulky probe I is sterically hindered to a larger degree than a bulky probe II for gaining access to the complementary sequence of a target ssDNA (hybridization site). It follows that it is more difficult for the bulky probe to gain access to the complementary sequence located closer to the center of the target DNA. As a result, the rate of the hybridization is reduced more greatly when the sequence is located in a site nearer to the center. In other words, a bulky probe I whose sequence is complementary to that of the hybridization site located between the 3501st and the 3551st nucleotides (called the “center”) of a target ssDNA is influenced more significantly from steric hindrance than a bulky probe II whose sequence is complementary to that of the hybridization site located between the 7199th and the 7249th nucleotides (called the “terminus”) of the target ssDNA, and hence, the hybridization rate of the bulky probe I is much smaller than that of the bulky probe II, as described above. Let us describe the hybridization reaction between a bulky probe (probe DNA tethered to a gold nanoparticle) and a target ssDNA in an aqueous solution by use of the following reaction scheme: the bulky probe and the target ssDNA in the solution collide with each other randomly in vain, unless an effective collision takes place in such a manner that more than five complementary nucleotides of the target ssDNA happen to meet a portion of a probe DNA tethered to the gold nanoparticle of the bulky probe. Once the effective collision occurs, a quasistable nucleation complex forms, in which a portion of the complementary region of the target ssDNA binds temporally to a portion of the probe DNA. Finally, the quasi-stable complex changes into a probe-target hybrid by zipping up the rest of the complementary region of the target ssDNA and probe DNA.
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Figure 5. Probability distribution, gN(r), of an end-to-end distance of N segments with N ) 20, 40, 94, 140, and 188. The dotted and the dashed lines represent g94(r) and g188(r), respectively. The inset shows an expanded view. The areas of the hatched and the diagonal regions correspond to GLb(94) and G2Lb(188), respectively.
Figure 4. Panel a shows a schematic diagram of a bulky probe, while panels b and c show illustrations of how DNA I and DNA II bind with the bulky probes, where a and b represent the diameters of a gold nanoparticle and a probe DNA, respectively and c and d represent the diameters of the bulky probe (Lb) and its access region, which is twice as large as the diameter (Lb) of the bulky probe. The solid squares in panels b and c represent the hybridization sites.
This reaction scheme explains that the hybridization reaction is influenced by steric hindrance of the target ssDNA in the collision step between the bulky probe and the target ssDNA; for instance, the bulky probe hybridizes readily with a target ssDNA having the hybridization site in a stretched form, as stated previously. A semiquantitative model on hybridization between the bulky probe and the target ssDNA mentioned in the following refers to an occurrence probability of an effective collision and does not refer to the other steps of the hybridization reaction, including nucleation complex formation and the zipping process, which is strongly correlated with chemical details of the bulky probe and the target ssDNA. Figure 4 illustrates pictures of a bulky probe (panel a), a target ssDNA hybridizing with several bulky probes I (panel b), and a target ssDNA hybridizing with several bulky probes II (panel c). Let us construct a semiquantitative model on the assumption that (1) probe DNAs and target ssDNAs are represented by a freely jointed chain (FJC) whose segment length is 1.5 nm,26 (2) the hybridization proceeds readily when an end-to-end distance of the corresponding hybridization site (consisting of N (
