364
Bioconjugate Chem. 2001, 12, 364−371
Nanostructured DNA-Protein Aggregates Consisting of Covalent Oligonucleotide-Streptavidin Conjugates Christof M. Niemeyer,*,† Michael Adler,† Song Gao,‡ and Lifeng Chi‡ Universita¨t Bremen, FB2 - UFT, Biotechnologie und Molekulare Genetik, Leobener Strasse, D-28359 Bremen, Germany, and Physikalisches Institut der Universita¨t Mu¨nster, Wilhelm-Klemm Strasse 10, D-48149 Mu¨nster, Germany . Received July 26, 2000; Revised Manuscript Received November 30, 2000
Covalent conjugates consisting of streptavidin and a 24-mer single-stranded DNA oligonucleotide have been oligomerized by cross-linking with a 5′,5′-bis-biotinylated 169-base-pair double-stranded DNA (dsDNA) fragment. The oligomeric conjugates formed have been analyzed by nondenaturing gel electrophoresis and scanning-force microscopy (SFM). The comparison of analogous oligomers, prepared from native STV and the bis-biotinylated dsDNA fragment, revealed that the covalent STVoligonucleotide hybrid conjugates self-assemble to generate oligomeric aggregates of significant smaller size, containing on average only about 2.5 times less dsDNA fragments per aggregate. Likely, this is a consequence of electrostatic or steric repulsion between the dsDNA and the single-stranded oligomer covalently attached to the hybrid, as indicated from control experiments. Nevertheless, the singlestranded oligonucleotide moiety within the oligomeric conjugates can be used as a selective molecular handle for further functionalization and manipulation. For instance, it was used for specific DNAdirected immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that macromolecules, such as STV and antibody molecules, which are tagged with the complementary oligonucleotide, specifically bind to the supramolecular DNA-STV oligomeric conjugates. This leads to a novel class of functional DNA-protein conjugates, suitable, for instance, as reagents in immuno-PCR or as building blocks in molecular nanotechnology.
INTRODUCTION
Molecular nanotechnology concerns the fabrication of well-defined nanoscaled structural and functional devices, using the “bottom-up” approach, in which small molecular building blocks self-assemble to form larger elements (1). Current advances in this area suggest that biological macromolecules are suitable components in the biomimetic synthesis of nanostructured elements and materials. For this purpose, DNA is a particularly powerful construction material (2-4). Due to its unique recognition capabilities, physicochemical stability, mechanical rigidity, and high precision processibility, DNA has extensively been used to fabricate nanostructured scaffolds (3) as well as for the selective positioning of proteins (5, 6), and metal and semiconductor nanoclusters (7-9). In addition, other biomimetic approaches to assemble inorganic nanoparticles have been reported which are based on the use of protein recognition systems, such as the bacterial surface layer proteins (10, 11) and the streptavidin-biotin system (6, 12). We have recently reported on the self-assembly of bisbiotinylated DNA 1 and the biotin-binding protein streptavidin (STV) 2 which leads to the formation of oligomeric DNA-protein networks 3 (Figure 1) (13). The nanostructured networks had been characterized using scanningforce microscopy (SFM). These studies revealed that the STV molecules predominantly function as a bivalent or trivalent linker between adjacent double-stranded DNA (dsDNA) molecules, despite the tetravalent binding capacity of STV for biotinylated ligands. As a conse* To whom correspondence should be addressed. Phone: (49) 421 218-4911; Fax: (49) 421 218-7578, e-mail:
[email protected]. † Universita ¨ t Bremen. ‡ Physikalisches Institut der Universita ¨ t Mu¨nster.
quence, the supramolecular DNA-STV networks 3 have a high residual biotin-binding capacity, and thus they can be employed as powerful reagents in Immuno-PCR (IPCR) (14), a method for the ultrasensitive detection of proteins and other antigens. In addition, the oligomeric networks 3 have also great potential for applications in biomolecular nanotechnology. For instance, the networks 3 can be used as a starting material for the synthesis of well-defined nanostructures, supramolecular DNA cycles consisting of a single STV and one dsDNA molecule (15). Moreover, the dsDNA-STV oligomers 3 are suitable model systems for basic studies on self-assembled nanoparticle networks and might even be used for the fabrication of ion-switchable nanoarchitecture (16). We here report on the incorporation of covalent oligonucleotide-STV conjugates 4 as distinct building blocks within oligomeric dsDNA-STV networks 5 (Figure 1). Since the coupling of biotinylated macromolecules occurs preferentially at terminal STV molecules of the oligomeric chains 3, and also, since some biotinylated compounds degrade the networks (13), we anticipated that the covalently attached oligonucleotide moiety in 4 might be a suitable molecular handle for the selective modification of oligomeric nanostructures 5. For instance, the coupling of other macromolecules tagged with a complementary oligonucleotide should be attainable. Thus, oligomeric aggregates 5 containing the covalent hybrids were assembled and characterized using native gelelectrophoresis and SFM. It was observed that the oligonucleotide-containing oligomeric aggregates 5 are significantly smaller in size than comparable aggregates 3 assembled from native STV. Nevertheless, the oligomers were found to be functional with respect to the hybridization of complementary nucleic acids. This allowed, for instance, the selective immobilization of 5 to capture oligonucleotide-modified surfaces, and the use of
10.1021/bc000090x CCC: $20.00 © 2001 American Chemical Society Published on Web 04/27/2001
DNA−STV Oligomers
Bioconjugate Chem., Vol. 12, No. 3, 2001 365
Figure 1. Synthesis of oligomeric streptavidin networks using 5′,5′-bis-biotinylated DNA spacers. (A) Formation of supramolecular networks consisting of a 169 bp dsDNA fragment 1 and native streptavidin (STV) 2. Note that the schematic drawing of the oligomers 3 is simplified, since branch-points occur due to the presence of tri- and tetravalently conjugated STV (see Figure 3). (B) Assembly of networks from the dsDNA 1 and covalent oligonucleotide-STV conjugates 4. The latter hybrid molecules were synthesized by covalent coupling of 5′-thiol-modified oligonucleotides and STV using a heterobispecific cross-linker (5). The assembly of 2 and 4 leads to the formation of oligomers 5 containing oligonucleotide moieties which can be used for functionalization of 5 by hybridization with complementary oligonucleotide-tagged macromolecules (see Figure 5). For simplification, complementary DNA strands are drawn as parallel lines. The 3′-ends are indicated by the arrowheads.
5 as a novel class of supramolecular reagents in IPCR applications. MATERIALS AND METHODS
Synthesis and purification of covalent DNA-STV hybrids, 4a and 4b, were carried out from the corresponding thiolated oligonucleotides, 5′-thiol-AGC GGA TAA CAA TTT CAC ACA GGA-3′ (4a), 5′-thiol-TCC TGT GTG AAA TTG TTA TCC GCT-3′ (4b), respectively, and recombinant streptavidin 2 (IBA, Go¨ttingen), similar as previously described (5). In brief, STV (10 nmol) was derivatized with maleimido groups using a heterobispecific cross-linker (sulfo-SMCC, Pierce), reacted with a 5′thiolated oligonucleotide (10 nmol) and subsequently the unreacted maleimido groups were quenched with an excess of mercaptoethanol. The crude product was purified by anion-exchange chromatography, and the one-toone molar ratio of the oligonucleotide and STV moiety within the hybrids was verified by gel-electrophoretic and photometric analysis (5). For control purposes, STV 2a was activated with the cross-linker but quenched with an excess of mercaptoethanol instead of the reaction with the thiolated oligonucleotide. The 169 base pair bis-biotinylated dsDNA fragment 1 was prepared from M13mp18 template (Promega) by preparative polymerase chain reaction (PCR), using two biotinylated primers: 5′-biotin-AGC GGA TAA CAA TTT CAC ACA GGA-3′ (bcA) and 5′-biotin-AAG GCG ATT AAG TTG GG-3′ (bG) as previously described (13). A monobiotinylated 169 bp dsDNA fragment 1a was synthesized for the preparation of model conjuages. An additional bisbiotinylated 256 bp dsDNA fragment was prepared from pUC19 template, similar as described in
reference (13). The dsDNA products were purified by gelfiltration chromatography, quantified by photometry and characterized by nondenaturing agarose gel electrophoresis in the presence of a DNA molecular weight standard. Oligomeric conjugates 3 and 5 were prepared from recombinant STV 2 or the DNA-STV hybrids 4, respectively. As an example, 1 µL of the dsDNA, 3 µM in 10 mM Tris buffer, pH 7.3 containing 5 mM EDTA (buffer A), was mixed with 14 µL of STV or hybrids 4 (0.2 µM in Buffer A), and the mixture was incubated typically for out about 24 h at 4 °C. The low ionic strength buffer A was chosen to avoid the condensation of the DNA fragments during immobilization on mica for SFM inspection (16). For gel electrophoretic analysis, the molar ratio of dsDNA to STV ranged from 4:1, 2:1, 1:1, 1:2, 1:4, to 1:10, respectively, as indicated in Figure 2. Samples for SFM imaging were prepared by placing a 6 µL drop of a solution containing 4 µM MgCl2 and about 500 nM DNA-STV conjugates on a Parafilm sheet. The droplet was adsorbed onto freshly cleaved mica and left to fix for 1 min. The drop was then quickly washed three times with 50 mL of deionized water and immediately blown dry for 5 min with nitrogen gas. The SFM inspection was carried out with commercial instruments (Digital Instrument, Dimension 3000 TM and Multimode III TM) using Si cantilevers purchased from Nanosensor TM. The SFM images were taken with instruments operating in high-amplitude dynamic mode with a homemade active feedback circuit (17), preventing the onset of intermitted contact (tapping). With this circuit, SFM can be run stable in the attractive interaction regime in air so that the interaction between the scanning tip and the sample is minimized.
366 Bioconjugate Chem., Vol. 12, No. 3, 2001
Niemeyer et al.
Figure 2. Gel electrophoretic analysis of the self-assembly of biotinylated dsDNA and streptavidin. (A) Formation of adducts from native streptavidin (STV) 2 and DNA-STV hybrid 4 with biotinylated dsDNA fragments. This is a nondenaturing 2% agarose gel stained with ethidium bromide. Adducts were formed by mixing bisbiotinylated dsDNA 1 with 1 or 4 mol equiv of native STV (lanes 9 and 10, respectively). The coupling of equimolar amounts of 1 and 2 leads to the formation of an immobile band (position k in lane 9), while in the case of the coupling of 1 and STV-hybrid 4a (lanes 4, 5), individual bands of lower molar weight are observed. As model compounds, adducts obtained from STV and varying amounts of monobiotinylated dsDNA 1a are analyzed in lanes 6-8. The analogous adducts obtained from hybrid 4a are shown in lanes 1-3. The small letters at the right side indicate the mobilities observed for free dsDNA 1 (a) and the adducts STV-DNA1 (b, f), STV2-DNA1 (c), STV-DNA2 (d, g), STV-DNA3 (e, h), and STV-DNA4 (i). Lane M contains a DNA molecular weight marker, the lengths are indicated as base pairs. The free conjugate 4a comigrates with the 396bp fragment of the molecular weight marker (not shown). (B) Comparison of native STV 2, chemically activated STV 2a, and DNA-STV hybrid 4a in the aggregation with bisbiotinylated dsDNA 1. This is a nondenaturing 1.5% agarose gel stained with ethidium bromide. A fingerprint-like band pattern of adducts is formed by mixture either 2, 2a, or 4a with different ratios of 1, ranging from 0.1 to 2 mol equiv of the DNA. Note the similarity of the patterns observed for 2 (lanes 9-12) and 2a (lanes 5-8), compared to the smaller products obtained from 4a (lanes 1-4). Some differences between 2a and 2 are visible in lanes 7 and 11. While native STV and an equimolar amount of 1 form an immobile product band (lane 11, see also position k in Figure 2a), this band is missing for the STV 2a (lane7), and also smaller aggregates are obtained in the case of an excess of the STV components 2 and 2a in lanes 9 and 5, respectively. In contrast, a completely different band pattern is obtained from 4a, indicating the formation of a dumbbell-shaped conjugate STV2-DNA1 as the major product (position c in Figure 2a).
DNA−STV Oligomers
Solid-phase hybridization of the conjugates 5 were carried out on STV-coated microplates, functionalized with a biotinylated capture oligonucleotide, complementary to 4a, similar as described earlier (18). A 30 µL volume of a 3.3 fM solution of 5 in buffer A was incubated in the wells of this microplate for 30 min at room temperature, and after washing, the immobilized DNA was amplified by PCR. The low ionic strength buffer A was chosen to allow for comparison with previous experiments. Quantification of the PCR products was carried out by microtiter-plate based enzyme-linked immunosorbent assay (19). For Immuno-PCR analysis, serial dilutions of rabbit IgG model antigen (Sigma, St. Louis, MO) immobilized in TopYield modules (Nunc) were used as a model antigen as previously reported (13, 19). For preparation of conjugates 6 and 7, 5 µL of a 500 nM solution of biotinylated goat anti-rabbit antibody (Coulter Immunotech, Hamburg, Germany) was either coupled with 15 µL of a 166 nM solution of 3, or else, similar amounts of the antibody were coupled with 10 µL of a 250 nM of 4b. In the latter case, the antibody-conjugated DNA-STV hybrid 4b was quenched with excess D-biotin and subsequently mixed with equimolar amounts of the D-biotin saturated oligomer 5. The mixture was incubated for 3 h at room temperature and then used in IPCR assay, as described (13). RESULTS AND DISCUSSION
Self-Assembly of the DNA-Streptavidin Complexes 3 and 5. A 169 bp bis-biotinylated dsDNA 1 was synthesized by preparative PCR using two 5′-biotin derivatized primer oligonucleotides, and, for control purposes, the analogous mono-biotinylated dsDNA fragment 1a was similarly prepared from one 5′-biotin derivatized and one unlabeled primer. Covalent hybrids of DNA and streptavidin, 4a and 4b, were synthesized from 5′-thiolated oligonucleotides and recombinant streptavidin by chemical cross-linking as previously described (5). The chief products, hybrids containing a single oligonucleotide moiety per STV, were purified by chromatography. Figure 2a shows the electrophoretic comparison of the adducts of monobiotinylated 1a and the STV hybrid 4a (lanes 1-3), or else, with native STV (lanes 6-8), respectively. Lanes 2 and 7 compare the conjugates obtained from the coupling of monobiotinylated dsDNA 1a with equimolar amounts of 4a or STV 2, respectively. The covalently linked oligonucleotide domain in 4a influences the electrophoretic mobility of the monoadducts, STV-DNA1 and 4a-DNA1, respectively (position b, f in Figure 2a). Moreover, it can be seen that the relative ratio of the mono- and bis-adducts is about 1:4 in the case of native STV, while in the case of the STV hybrid 4a, this ratio is only about 5:1. This suggests that the affinity of 4a for the biotinylated dsDNA 1a is decreased compared to the native STV. The comparison of lanes 2 and 7 in Figure 2a supports this assumption. Native STV forms adducts of a relative stoichiometry of STV-DNA3,4 to a small but significant extent. The preferential formation of bis-adducts has previously been observed (13). However, in the case of the hybrid 4a (lane 2), the analogous complex, 4a-DNA4 is entirely missing. The reduced affinity of 4a for the biotinylated dsDNA can also be deduced from the comparison of lanes 4 and 9 (Figure 2a), in which the bis-biotinylated dsDNA 1 was mixed with equimolar amounts of STV 2 or 4a, respectively. In the case of native STV, this coupling leads to
Bioconjugate Chem., Vol. 12, No. 3, 2001 367
the formation of a single immobile band (position k), indicating the formation of large oligomeric networks 3 in lane 9. In contrast, the coupling of 4a leads to the formation of individual bands, representing the presence of small supramolecular aggregates 5 (lane 4). The reduced affinity of 4a for the biotinylated dsDNA might be due to an increased steric or electrostatic repulsion of the dsDNA and the covalently bound oligonucleotide, or else, due to a decreased biotin-binding affinity itself, possibly caused by a conformational change induced by the chemical treatment of the STV's surface. The latter might be possible despite the fact that we had previously established the complete tetravalent binding capacity of the covalent hybrids 4 for the binding of low molecular weigth D-biotin (5). To further elucidate why only small aggregates are formed from the hybrids 4, controls were carried out with STV 2a, activated with the cross-linker but not exposed to the thiolated oligonucleotide (Figure 2b). The coupling of various molar equivalents of the bisbiotinylated dsDNA 1 revealed an almost unchanged capability of the control STV 2a for binding the biotinylated dsDNA, as compared to either the native STV 2 or the DNA-STV hybrid 4a. These results suggest that the decrease in oligomer formation of 4a is mainly due to steric and/or electrostatic repulsion of the dsDNA and the single-stranded oligomer. It seems likely that an increase in the ionic strength of the buffer would compensate for electrostatic repulsion. However, we have used a low salt 10 mM Tris buffer throughout this initial study for two reasons. First, these conditions are equal to that of a previous study, thereby allowing for a direct comparison of the results (13). Second, the use of higher salt concentration, in particular the presence of magnesium, leads to the formation of anomalous aggregate structures (16). In particular, a condensation of the DNA fragments occurs which makes the aggregates difficult to analyze by SFM. Nevertheless, we anticipate that the detailed optimization of coupling conditions, such as a systematic variation of polyvalent cations and ionic strengths, will lead to novel means of affecting the aggregate’s supramolecular structure. To study the structures of the oligomeric DNA-STV complexes, samples of 3 and 5 were imaged using dynamic SFM working in attractive regime mode. Representative images are shown in Figure 3, and statistical analysis of the complexes observed are given in Table 1. As expected from the gel electrophoretic characterization (Figure 2), the average size of 3 is more than 2-fold greater than that of 5. On average, the oligomers 5 contain only about 2.0 dsDNA fragments per supramolecular conjugate. Even more significant, however, is the complete lack of highly oligomerized networks, containing more than 10 DNA molecules per conjugate (Table 1). Moreover, the comparison of the streptavidin’s valency reveals that in the case of the oligomers 5, on average, only 1.46 dsDNA fragments are coupled per protein particle. In contrast, an average STV valency of 2.4 is observed in the oligomers 3. Experiments carried out with DNA-STV hybrids containing other sequences of the single-stranded oligomer, such as 4b, led to similar aggregation patterns, as judged from gel-electrophoretic analysis (data not shown). These observations again indicate the decreased affinity of 4 for the binding of biotinylated dsDNA, as compared to native STV 2. Functionality of the Oligonucleotide Domain of Oligomers 5. As an initial test of the functionality of the oligonucleotide domain of 5, the supramolecular aggregates 5 were hybridized to complementary capture oligonucleotides, previously immobilized in STV-coated
368 Bioconjugate Chem., Vol. 12, No. 3, 2001
Niemeyer et al.
Figure 3. Scanning force microscopy images of oligomeric dsDNA-STV conjugates. (A) DNA-STV networks 3 obtained from native STV. (B) Oligomers 5 obtained from dsDNA 1 and covalent oligonucleotide-STV conjugates 4. (C) A preformed oligomer 5 was coupled with the complementary DNA-STV hybrid 4b. The arrows indicate the occurrence of 4a‚4b dimers, formed by hybridization of the two complementary oligonucleotide moieties. (D) Interconnected networks obtained from the self-assembly of two complementary STV hybrids 4a and 4b, subsequently oligomerized using a bis-biotinylated 256 bp dsDNA. Note that these nanoparticle networks contain two different dsDNA spacers, the ca. 87 nm dsDNA and the about 7 nm dsDNA formed by the hybridization of 4a and 4b (arrows). The latter dsDNA spacer fragment, however, cannot directly be observed by SFM imaging. Table 1. Statistical Analysis of SFM Images of Oligomeric Complexes 3 and 5 Obtained from dsDNA 1 and Native STV 2 or STV Hybrid 4a native conjugates hybrid conjugates dsDNA‚2 dsDNA‚4a size distribution [%]b no. of DNA/complex 1 2 3 4 5-10 >10 sizemax [DNA/complex]c sizeavg [DNA/complex]d valency of STV [%]e monovalent STV divalent STV trivalent STV tetravalent STV avg valency [dsDNA/STV]
26 36 19 7 9 2 30 5
49 29 13 3 5
