Thermoprecipitation of Streptavidin via Oligonucleotide-Mediated Self

22, 5530r9]. In the work presented here, streptavidin was noncovalently coupled with .... night with 10 μequiv of DEAE Sepharose fast anion- exchange...
0 downloads 0 Views 105KB Size
Download PDF
720

Bioconjugate Chem. 1999, 10, 720−725

Thermoprecipitation of Streptavidin via Oligonucleotide-Mediated Self-Assembly with Poly(N-isopropylacrylamide) Robin B. Fong, Zhongli Ding, Cynthia J. Long, Allan S. Hoffman,* and Patrick S. Stayton* Department of Bioengineering, University of Washington, Seattle, Washington 98195

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquidphase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 °C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.

INTRODUCTION

Affinity chromatography techniques which utilize matrix-immobilized ligands are the most widely used methods for specific isolation of molecules in separations and diagnostic technologies (1). There has been growing interest in developing liquid-phase separation techniques such as affinity partitioning and precipitation, which typically involve ligand-bearing polymers that can be phase separated after binding to complementary biomolecules (2-6). In affinity precipitation, the phase separation of polymer and polymer-bound species can be triggered by a small change in an environmental condition, such as pH, ionic strength, or temperature. Stimuliresponsive polymers have been employed in many applications in medicine and biotechnology (7, 8). One of the best examples is poly(NIPAAM),1 a water-soluble, synthetic polymer which undergoes a phase transition to a hydrophobic state when the temperature is raised above its lower critical solution temperature (LCST) (9). Poly(NIPAAM) has been conjugated to immunoglobulin G, β-D-glucosidase, and trypsin to enable thermoprecipitation of these proteins (10-13). These alternative systems circumvent some of the shortcomings of chromatographic methods, because the ligand association step occurs in the solution phase. These systems exhibit fewer mass transport and steric limitations which exist with solid supports. * To whom correspondence should be addressed. (A.S.H.) Department of Bioengineering, Box 352255. Phone: (206) 543-9423. Fax: (206) 543-6124. E-mail: hoffman@ u.washington.edu. (P.S.S.) Department of Bioengineering, Box 352125. Phone: (206) 685-8148. Fax: (206) 685-8256. E-mail: [email protected].

Another potential advantage of affinity precipitation strategies is the ability to selectively and sequentially separate different targets from complex mixtures. Polymers with responses that have been engineered for different temperature or pH ranges can be conjugated to different ligands and mixed together in order to sequentially pull targets out of solution under different conditions. Here we report a versatile strategy for sequentially separating selective components from complex mixtures using stimuli-responsive polymers and streptavidin. Streptavidin is a stable, tetrameric protein that binds as many as four biotin molecules with extraordinary affinity and is utilized widely in current separations and diagnostic applications (14-23). To reversibly conjugate poly(NIPAAM) to streptavidin, we have used a recently described strategy for selectively joining two molecular species through the self-assembly of complementary oligonucleotides. Niemeyer et al. showed that oligonucleotides can direct the assembly of bispecific and bifunctional supramolecular structures, and they used this clever strategy to make streptavidin conjugates 1 Abbreviations: poly(NIPAAM), poly(N-isopropylacrylamide); LCST, lower critical solution temperature; STAV, streptavidin; STAV-nR, streptavidin conjugated to n number of oligonucleotide R; s-SMCC, sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; TCEP-HCl, Tris(2-carboxyethyl)phosphine hydrochloride; NHS, N-hydroxysuccinimidyl; DEAE, diethylaminoethyl; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetracetic acid; DMF, dimethylformamide; PB75, 100 mM phosphate and 100 mM NaCl, pH 7.5, buffer; TB75, 10 mM Tris and 1 mM EDTA, pH 7.5, buffer; TB74, 50 mM Tris, 150 mM NaCl, and 0.05% bovine serum albumin, pH 7.4, buffer; BAP, biotinylated alkaline phosphatase; RT, room temperature.

10.1021/bc980151f CCC: $18.00 © 1999 American Chemical Society Published on Web 08/25/1999

Thermoprecipitation of Streptavidin

Figure 1. Schematic representation of reversible hybridization of a streptavidin-oligonucleotide conjugate and a temperature responsive polymer-oligonucleotide conjugate, and reversible phase separation of the resultant complex in response to a small temperature change.

(24, 25). Figure 1 illustrates the case of reversible selfassembly of streptavidin and a temperature-responsive polymer, followed by reversible phase separation of the complex. Because the assembly is DNA sequence specific, polymers with different stimuli responsiveness can be selectively attached to streptavidin conjugates that are similarly differentiated by their DNA sequences. Thus, different streptavidin-target complexes can be selectively and sequentially separated from a complex liquid mixture depending on which polymer response is triggered. EXPERIMENTAL PROCEDURES

Materials. Oligodeoxyribonucleotide R (5′-GGACTCAGGCTTATAGCTGT-3′), modified at the 5′ end with a 6-carbon aliphatic amine, and complementary oligodeoxyribonucleotide RC (5′-ACAGCTATAAGCCTGAGTCC3′), modified at the 3′ end with a 7-carbon aliphatic amine, were purchased from Integrated DNA Technologies (Coralville, IA). A melting temperature of ca. 65 °C for the renatured duplex R:RC (in 50 mM Tris and 150 mM NaCl, pH 7.4) was measured spectrophotometrically in our laboratory. Oligodeoxyribonucleotide S (5′-AGCGGATAACAATTTCACAC-3′), which shares no sequence complementarity with oligo R or RC, was used as a control sequence. The heterobifunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (s-SMCC) and the disulfide reducing agent Tris(2-carboxyethyl) phosphine hydrochloride (TCEPHCl) were obtained from Pierce (Rockford, IL). Trisglycine and Tris-borate-EDTA-urea polyacrylamide gels and gel reagents were obtained from Novex (San Diego, CA). Sephadex gel (G25-medium and G50-fine) and DEAE Sepharose fast anion-exchange resin were from Pharmacia Biotech (Uppsala, Sweden). Centricon and Centriprep ultrafiltration concentrators were purchased from Amicon (Beverly, MA). D-[8,9-3H]Biotin was from Amersham (Little Chalfont, England), and EcoLume liquid scintillation cocktail was from ICN (Costa Mesa, CA). Biotinylated calf intestine alkaline phosphatase was from Pierce, and p-nitrophenyl phosphate and diethanolamine were from Bio-Rad (Hercules, CA). Streptavidin Production. The gene for streptavidin mutant E116C (cysteine in place of glutamic acid at amino acid position 116) was created by cassette mutagenesis, and the protein was expressed, purified, and characterized using previously described protocols (26). Polymer Synthesis. Linear poly(NIPAAM), with one end having an N-hydroxysuccinimidyl (NHS) ester group, was produced according to the methods of Ding et al. (13).

Bioconjugate Chem., Vol. 10, No. 5, 1999 721

For the polymer used in this study, a mean molecular weight of 8500 was determined by vapor pressure osmometry (VPO, model OSV111, Knauer, Germany). Streptavidin-Oligonucleotide Conjugation. Oligonucleotide R (∼200 nmol) was derivatized with a maleimido group by reacting it with 50-fold molar excess (4.2 mg) of s-SMCC in 1 mL of 100 mM phosphate and 100 mM NaCl, pH 7.5 (PB75). The reaction was incubated for 1 h at RT. Excess s-SMCC was removed using a Sephadex G25-medium gel filtration column (1.5 × 7 cm). Streptavidin mutant E116C (∼600 nmol) in 2 mL of PB75 was reduced by addition of 150 µL of 40 mM TCEP-HCl. The amount of streptavidin was determined using an extinction coefficient of 34 000 M-1 cm-1 for the monomer subunit (27). After 15 min at RT, the gel filtration fractions containing maleimide-derivatized oligonucleotide (1.5 mL total) were added. The reaction proceeded for 2 h at RT with a 50 µL addition of freshly made 40 mM TCEP-HCl at 1 h. Excess oligonucleotide was removed using Centricon-30 (30 000 MW cutoff) concentrators. The remaining unreacted oligonucleotide was removed using a Sephadex G50-fine gel filtration column (1.0 × 16 cm). Streptavidin conjugate was purified from unreacted streptavidin by anion-exchange perfusion chromatography using a BioCAD system (PerSeptive Biosystems, Farmington, MA). The sample was applied to a Poros HQ/M (PerSeptive Biosystems) 29 mL column and eluted with a salt gradient of 0.4 to 1.0 M NaCl in 20 mM Tris, pH 6.8. The fractions containing conjugate were concentrated using Centriprep-30 concentrators and analyzed by 10-20% Tris-glycine SDSPAGE. Poly(NIPAAM)-Oligonucleotide Conjugation. Oligonucleotide RC (∼1.5 µmol) was dissolved in 7.2 mL of 100 mM borate, 50 mM NaCl, and 5 mM EDTA, pH 9.5. A 50-fold molar excess of NHS ester-activated poly(NIPAAM) (96 mg) was dissolved in 800 µL of HPLCgrade DMF and added to the oligonucleotide solution. The reaction was gently mixed for 4 h at 4 °C, followed by 2 h at RT. To separate polymer from unreacted oligonucleotide, 100 µL of 5 M NaCl was added (to lower the LCST), and the temperature was raised to 37 °C, precipitating the polymer and conjugate. After 29000g centrifugation at 37 °C, the supernatant was decanted. The pellet was dissolved overnight in 4 mL of 10 mM Tris and 1 mM EDTA, pH 7.5 (TB75) at 4 °C, and the separation process was repeated twice. Polymer-oligonucleotide conjugate was purified from excess polymer by capturing it overnight with 10 µequiv of DEAE Sepharose fast anionexchange resin at 4 °C in 4 mL of TB75. The resin-bound conjugate was washed three times with TB75 at 4 °C and eluted with 0.7 M NaCl. The conjugate was analyzed using 15% Tris-borate-EDTA-urea PAGE. Precipitation of Biotin and Biotinylated Alkaline Phosphatase. Tritiated biotin (1.8 pmol) was incubated with an equimolar amount of STAV-1R (streptavidin with one attached oligonucleotide) and a 20-fold molar excess amount of poly(NIPAAM)-RC conjugate in 50 mM Tris, 150 mM NaCl, and 0.05% BSA, pH 7.4, buffer (TB74). Final concentrations of STAV-1R and poly(NIPAAM)-RC were 18 and 360 nM, respectively. After reaction for 1 h at RT, free poly(NIPAAM) (1 mg/mL final concentration) was added, and the sample was placed in a 37 °C bath for 10 min and then centrifuged at 16000g for 2 min. (Addition of the unmodified free polymer helped to enhance the precipitation.) The amount of biotin in the supernatant was measured by β-radiation counting (Beckman LS7000, Irvine, CA) using EcoLume scintillation cocktail. To show that hybridization was

722 Bioconjugate Chem., Vol. 10, No. 5, 1999

occurring, oligonucleotides R, RC, or S were individually added at final concentrations ranging from 10-3 to 104 times that of STAV-1R. Recovery of pelleted STAV-1R (and bound biotin) was accomplished by overnight dissolution of the pellet in deionized water at 4 °C, followed by heating and centrifugation as described earlier. Biotinylated alkaline phosphatase (BAP) and poly(NIPAAM)-RC were added to STAV-1R at a 1:20:400 molar ratio BAP:STAV-1R:poly(NIPAAM)-RC in TB74. Final concentrations of STAV-1R and poly(NIPAAM)RC were 12 and 240 nM, respectively. The reaction was allowed to incubate for 1 h at RT. After addition of free poly(NIPAAM) (1 mg/mL final concentration), the sample was placed in a 37 °C bath for 10 min and then centrifuged at 16000g for 2 min. The supernatant activity was measured by reaction with p-nitrophenyl phosphate substrate in diethanolamine and UV absorbance at 405 nm. Cloud Point Measurement. Cloud point data for poly(NIPAAM)-oligonucleotide conjugate, STAV/DNA/ poly(NIPAAM) complex, and pure poly(NIPAAM) were taken using a UV-vis spectrophotometer with a jacketed cuvette holder. The heating rate was 0.2 °C/min. Polymer solutions were prepared by dissolving 8500 molecular weight poly(NIPAAM) in TB74 buffer at 0.005, 0.003, and 0.002 wt % polymer concentrations. The sample containing poly(NIPAAM)-RC (5 µM) was also prepared in TB74. The sample containing STAV/DNA/polymer complex (5 µM) was prepared by reacting equimolar quantities of STAV-1R conjugate and poly(NIPAAM)-RC conjugate in TB74 for 1 h at RT. The bioconjugate solutions were estimated to contain ca. 0.004 wt % poly(NIPAAM), assuming a molecular weight of 8500 for the conjugated polymer. RESULTS

Preparation of Conjugates. Anion-exchange chromatography and PAGE analysis of the purified conjugation reaction products revealed three major species (Figure 2). Peaks I and II (combined in lanes 2 and 7) display only protein bands, and thus represent STAV and multimers of STAV. The bands migrated equivalently to a control sample of STAV (lane 1). Boiled samples displayed protein bands that were the correct size for streptavidin monomer subunits (lane 5). Peaks III and IV, however, displayed bands that stained positive for both protein (lanes 3 and 4, respectively) and DNA (lanes 8 and 9, respectively). These bands migrated faster than the STAV control, consistent with the increase in negative charge with the addition of a conjugated oligonucleotide. Because peak IV species migrated farther than peak III and because peak IV eluted off the cationic column under higher salt concentration compared to peak III, it is likely that peak IV represents a more highly conjugated STAV than peak III. Peak IV likely represents STAV with two oligonucleotide attachments (STAV-2R) and peak III represents STAV with one oligonucleotide attachment (STAV-1R). Secondary bands are oligomeric species which escaped disulfide reduction, e.g., STAV dimer conjugated to one oligonucleotide, or STAV dimer with a single oligonucleotide conjugated to each STAV. Figure 3 demonstrated that the polymer-oligonucleotide conjugation reaction produced covalent poly(NIPAAM)-RC conjugates. After thermal precipitation, which removed excess oligonucleotide, the pellet was redissolved and subjected to anion exchange using DEAEmodified Sepharose beads. Poly(NIPAAM)-RC conjugate

Fong et al.

Figure 2. (a) Anion-exchange chromatography of the streptavidin-oligonucleotide conjugation reaction mixture on a PerSeptive Biosystems Poros HQ/M analytical column (4.6 × 100 mm, 1.7 mL). Four absorbance peaks (I-IV) were observed. The beginning of the chromatogram is magnified (inset) to show peaks I and II. (b) Electrophoretic analysis of the streptavidinoligonucleotide conjugate chromatogram peaks using a 10-20% Tris-glycine-SDS polyacrylamide gel. Arrow indicates direction of migration. Samples were reduced by addition of β-mercaptoethanol to 5% volume. The gel on the left was stained for protein using Coomassie blue dye while the gel on the right was stained for DNA using ethidium bromide. Lanes 1 and 10: STAV control. Lanes 2 and 7: peaks I and II combined (STAV and multimers). Lane 5: peaks I and II combined, heated for 10 min at 99 °C to dissociate streptavidin into monomers. Lanes 3 and 8: peak III (STAV-1R conjugate). Lanes 4 and 9: peak IV (STAV-2R conjugate). Lane 6: Novex Multimark protein standard.

was expected to bind to the positively charged DEAE, while excess polymer remained in solution. The eluted product stained positive for DNA (lane 3), and gel migration was hindered compared to a control which contained oligonucleotide RC alone (lane 1). Free polymer did not impede migration of unmodified oligonucleotide (lane 2). Precipitation of Biotin and Biotinylated Alkaline Phosphatase. Panels a and b of Figure 4 summarize the results of the [3H]biotin and biotinylated alkaline phosphatase (BAP) separations, respectively. A control without poly(NIPAAM)-RC was included in each experiment to rule out nonspecific precipitation. The activity measurements were normalized against that of an internal reference composed of [3H]biotin (or BAP) and STAV-1R in the same buffer, without free polymer. When poly(NIPAAM)-RC and STAV-1R were both present, most of the [3H]biotin (∼76%) and BAP (∼85%) were removed from solution after heating. The pelleted [3H]biotin (and STAV-1R) could be subsequently redissolved

Thermoprecipitation of Streptavidin

Figure 3. Electrophoretic analysis of the poly(NIPAAM)oligonucleotide conjugation reaction using an ethidium bromidestained 15% Tris-borate-EDTA-urea polyacrylamide gel. Arrow indicates direction of migration. Lane 1: oligonucleotide RC control. Lane 2: physical mixture of oligonucleotide RC and free poly(NIPAAM) (∼1 mg/mL). Lane 3: poly(NIPAAM)-RC conjugate.

Bioconjugate Chem., Vol. 10, No. 5, 1999 723

Figure 5. Free oligonucleotide inhibition curves for the thermally induced affinity precipitation of [3H]biotin. Experiments were performed by adding varying amounts of a free oligonucleotide (R, RC, or S) to a solution containing a fixed amount of [3H]biotin, STAV-1R, and poly(NIPAAM)-RC (molar ratio 1:1:20, respectively). The solution was allowed to react for 1 h at RT, prior to thermal precipitation. Oligo R is complementary to the sequence of RC; oligo RC is complementary to the sequence of R; and oligo S is not complementary to either R or RC. The vertical axis represents the amount (β count) of [3H]biotin remaining in the solution phase after precipitation. Measurements were normalized against a reference sample (see Results). Data are the average of three different experiments. The error bars indicate standard deviations.

Figure 4. (a) Thermally induced affinity precipitation and recovery of [3H]biotin. The solid bars represent the amount of [3H]biotin in solution measured by β counting. (b) Thermally induced affinity precipitation of biotinylated alkaline phosphatase (BAP). The solid bars represent the BAP enzymatic activity in solution measured by colorimetric assay. Measurements were normalized against an appropriate reference sample (see Results). Data are the average of three different experiments. The error bars indicate standard deviations.

when the pellet was chilled in deionized water. STAV1R is presumed to be separated from poly(NIPAAM)RC under these conditions, which favor denaturation of the 20 base pair DNA. The sequence-specificity of the self-assembling DNA was tested by addition of competing and noncompeting oligonucleotides in the [3H]biotin experiment. In Figure 5, the free oligo RC begins to inhibit the precipitation of labeled biotin at a concentration equimolar to that of STAV-1R. Free oligo R inhibits the precipitation when its concentration is about 20 times that of STAV-1R. The inhibition curve for oligo R is shifted by a factor of about 20 relative to the curve for oligo RC, consistent with the 20:1 molar ratio of polymer-RC conjugate to STAV-1R conjugate in each of the experiments. Oligo S, which is not complementary in sequence to either R or RC, did not affect the precipitation. The results of this inhibition study indicate that a sequence-specific oligonucleotide

Figure 6. Cloud point curves for solutions containing 5 µM poly(NIPAAM)-RC (open squares); 5 µM STAV/DNA/poly(NIPAAM) complex (darkened squares); and 0.005 wt % (open triangles), 0.003 wt % (open circles), and 0.002 wt % (open diamonds) pure poly(NIPAAM).

hybridization reaction occurs prior to thermal precipitation of the streptavidin-biotin complex. Cloud Point Measurement. The reversible phase separation exhibited by poly(NIPAAM) as the solution temperature is raised above its LCST is characterized by an abrupt flocculation of the insoluble polymer chains (28). The thermally induced clouding of solutions containing either the poly(NIPAAM)-oligonucleotide conjugate or the STAV/DNA/poly(NIPAAM) complex was investigated and compared to solutions containing comparable concentrations of pure poly(NIPAAM) (Figure 6). The data demonstrate that the soluble-to-insoluble phase

724 Bioconjugate Chem., Vol. 10, No. 5, 1999

Fong et al.

Figure 7. Proposed application of self-assembled streptavidin-DNA-polymer conjugates for sequential affinity precipitation of different target species. A complex composed of a pH responsive polymer, streptavidin, and an antibody against target 1 (open square) is mixed with a complex composed of a temperature responsive polymer, streptavidin, and an antibody against target 2 (open triangle). After binding to their respective ligand targets, the complexes can be sequentially retrieved from solution. Target 1 is coprecipitated with the pH responsive complex via a small pH change. Afterward, target 2 is coprecipitated with the temperature responsive complex via a small temperature change. A third target (open circle) does not precipitate because it shares no affinity with either complex.

transition of the polymer was not significantly affected by the conjugated biomolecules. DISCUSSION

Chen and Hoffman introduced the concept of “smart” polymer affinity precipitation by using poly(NIPAAM)protein A conjugates to purify IgG antibodies (29). Others have shown that DNA-binding proteins can be precipitated on the basis of their affinity to poly(NIPAAM)DNA conjugates (30, 31). Related work has suggested that coprecipitation of hybridized poly(NIPAAM)-oligo(dT) conjugates may be a more efficient method for isolating polyadenylated mRNAs than conventional oligo(dT)-modified silica (32). While these strategies offer the advantages of liquid-phase binding steps, there are a number of parameters which require tailoring. For example, the activity of the conjugate may be affected depending on the number of polymer chains per biomolecule (33-36) and the location of these chains (37). In the present work, we have developed a self-assembly system by which a defined polymer and a defined affinity connector, such as a biotinylated antibody, can be joined together from a mixture of polymers, connectors, and targets by using a strategy described by Niemeyer et al. to construct intermolecular linkages. Distinct target species can be separated in solution through either a direct interaction with a biotin group on the target or through a secondary interaction with a biotinylated connector. The new feature represented in this approach is the capability to sequentially separate a specific targetbound complex from a mixture. The specific stimuliresponsive polymer can be matched to a unique affinity complex, e.g., a streptavidin-antibody conjugate, by selfassembly in solution. The use of DNA tags on streptavidin and the polymer ensures conjugation of the appropriate smart polymer to the target-bound complex. For example, in step 1 a small pH change can be used to separate pH responsive polymer/antibody1/target1 complexes, while in step 2 a small temperature change can be used to separate temperature responsive polymer/ antibody 2/target 2 complexes (Figure 7). To demonstrate this technique, we have constructed a poly(NIPAAM)/DNA/streptavidin complex that self-assembles and binds biotinylated alkaline phosphatase. The

entire assembly, including alkaline phosphatase (∼140 kDa), precipitates above the LCST of poly(NIPAAM) with high efficiency. Using radiolabeled biotin, we showed that the phase-separated streptavidin assembly could be fully resolubilized by lowering the temperature below the LCST of the poly(NIPAAM). If this is done under low salt conditions, the short double-stranded DNA melts, freeing the poly(NIPAAM)-DNA conjugate for recovery and recycling. Free oligonucleotides that were complementary to either the DNA tag on the streptavidin conjugate or the DNA tag on the poly(NIPAAM) conjugate could inhibit the affinity precipitation of radiolabeled biotin at stoichiometric ratios, whereas a random oligonucleotide had no effect. Bispecific streptavidin-oligonucleotide conjugates may offer other possibilities for novel applications involving stimuli-responsive polymers. For example, we have shown that poly(NIPAAM) can reversibly manipulate the biotinbinding properties of streptavidin when it is site specifically attached near the binding pocket (26, 38, 39). A potential advantage of using oligonucleotide linkers is the ability to control the polymer attachment distance by ratcheting the site where the DNA sequence overlap is located. The use of polymer-oligonucleotide conjugates may also provide a convenient method for sequentially directing the adsorption of biomolecules to a surface. For the probing of genes, it may be possible to add smart polymer function to DNA targets using a pH responsive polymer-oligonucleotide conjugate that remains soluble under the thermal denaturation conditions needed to prepare the target for hybridization with the probe. ACKNOWLEDGMENT

This work was supported by NIH Grant R01GM53771A, the Washington Technology Center, the Washington Research Foundation, and the Office of Technology Transfer. We gratefully acknowledge discussions with the Cantor group at Boston University for the initial suggestion of using oligonucleotides to assemble proteins and smart polymers. LITERATURE CITED (1) Jones, C., Patel, A., Griffin, S., Martin, J., Young, P., O’Donnell, K., Silverman, C., Porter, T., and Chaiken, I.

Thermoprecipitation of Streptavidin (1995) Current trends in molecular recognition and bioseparation. J. Chromatogr. A 707, 3-22. (2) Diamond, A. D., and Hsu, J. T. (1992) Aqueous Two-Phase Systems for Biomolecule Separation. In Advances in Biochemical Engineering/Biotechnology (A. Feichter, Ed.) Vol. 47, pp 89-135, Springer-Verlag, Berlin. (3) Luong, J. H. T., and Nguyen, A. L. (1992) Novel Separations Based on Affinity Interactions. In Advances in Biochemical Engineering/Biotechnology (A. Feichter, Ed.) Vol. 47, pp 13758, Springer-Verlag, Berlin. (4) Niederauer, M. Q., and Glatz, C. E. (1992) Selective Precipitation. Advances in Biochemical Engineering/Biotechnology (A. Feichter, Ed.) Vol. 47, pp 159-88, Springer-Verlag, Berlin. (5) Irwin, J. A., and Tipton, K. F. (1995) Affinity precipitation: a novel approach to protein purification. Essays Biochem. 29, 137-56. (6) Gupta, M. N., Kaul, R., Guoqiang, D., Dissing, U., and Mattiasson, B. (1996) Affinity precipitation of proteins. J. Mol. Recognit. 9, 356-9. (7) Hoffman, A. S. (1995) “Intelligent” Polymers in Medicine and Biotechnology. Macromol. Symp. 98, 645-64. (8) Galaev, I. Y., and Mattiasson, B. (1993) Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology. Enzyme Microb. Technol. 15, 354-66. (9) Heskins, M., and Guillet, J. E. (1968) Solution Properties of Poly(N-isopropylacrylamide). J. Macromol. Sci.-Chem. A2, 1441-55. (10) Monji, N., and Hoffman, A. S. (1987) A novel immunoassay system and bioseparation process based on thermal phase separating polymers. Appl. Biochem. Biotech. 14, 107-20. (11) Takei, Y. G., Matsukata, M., Aoki, T., Sanui, K., Ogata, N., Kikuchi, A., Sakurai, Y., and Okano, T. (1994) Temperature-Responsive Bioconjugates. 3. Antibody-Poly(N-isopropylacrylamide) Conjugates for Temperature-Modulated Precipitations and Affinity Bioseparations. Bioconjugate Chem. 5, 577-82. (12) Chen, G., and Hoffman, A. S. (1993) Preparation and properties of thermoreversible, phase-separating enzymeoligo(N-isopropylacrylamide) conjugates. Bioconjugate Chem. 4, 509-14. (13) Ding, Z., Chen, G., and Hoffman, A. S. (1996) Synthesis and purification of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. Bioconjugate Chem. 7, 121-6. (14) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85133. (15) Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184, 51-67. (16) Wilchek, M., and Bayer, E. A. (1990) Introduction to avidin-biotin technology. Methods Enzymol. 184, 5-13. (17) Wilchek, M., and Bayer, E. A. (1990) Applications of avidin-biotin technology: literature survey. Methods Enzymol. 184, 14-45. (18) Diamandis, E. P., and Christopoulos, T. K. (1991) The biotin-(strept)avidin system: principles and applications in biotechnology. Clin. Chem. 37, 625-36. (19) Bayer, E. A., and Wilchek, M. (1990) Application of avidinbiotin technology to affinity-based separations. J. Chromatogr. 510, 3-11. (20) Wilchek, M., and Bayer, E. A. (1990) Biotin-containing reagents. Methods Enzymol. 184, 123-38. (21) Bayer, E. A., and Wilchek, M. (1990) Protein biotinylation. Methods. Enzymol. 184, 138-60. (22) O’Shannessy, D. J. (1990) Antibodies biotinylated via sugar moieties. Methods Enzymol. 184, 162-6. (23) Klevan, L., and Gebeyehu, G. (1990) Biotinylated nucleotides for labeling and detecting DNA. Methods Enzymol. 184, 561-77.

Bioconjugate Chem., Vol. 10, No. 5, 1999 725 (24) Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA-streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 22, 5530-9. (25) Niemeyer, C. M., Burger, W., and Hoedemakers, R. M. (1998) Hybridization characteristics of biomolecular adaptors, covalent DNA-streptavidin conjugates. Bioconjugate Chem. 9, 168-75. (26) Chilkoti, A., Tan, P. H., and Stayton, P. S. (1995) Sitedirected mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc. Natl. Acad. Sci. U.S.A. 92, 1754-8. (27) Sano, T., and Cantor, C. R. (1990) Expression of a cloned streptavidin gene in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 87, 142-6. (28) Chen, J. P., Yang, H. J., and Hoffman, A. S. (1990) Polymer-protein conjugates I. Effect of protein conjugation on the cloud point of poly(N-isopropylacrylamide). Biomaterials 11, 625-30. (29) Chen, J. P., and Hoffman, A. S. (1990) Polymer-protein conjugates. II. Affinity precipitation separation of human immunogammaglobulin by a poly(N-isopropylacrylamide)protein A conjugate. Biomaterials 11, 631-4. (30) Maeda, M., Nishimura, C., Umeno, D., and Takagi, M. (1993) Modification of DNA with poly(N-isopropylacrylamide) for thermally induced affinity separation. Reactive Polym. 21, 27-35. (31) Umeno, D., and Maeda, M. (1997) Poly(N-isopropylacrylamide) Carrying Double-Stranded DNA for Affinity Separation of Genotoxins. Anal. Sci. 13, 553-6. (32) Umeno, D., Mori, T., and Maeda, M. (1998) Single stranded DNA-poly(N-isopropylacrylamide) conjugate for affinity separation of oligonucleotides. Chem. Commun. 1433-4. (33) Park, T. G., and Hoffman, A. S. (1993) Synthesis and characterization of a soluble, temperature-sensitive polymerconjugated enzyme. J. Biomater. Sci. Polym. Ed. 4, 493-504. (34) Matsukata, M., Takei, Y., Aoki, T., Sanui, K., Ogata, N., Sakurai, Y., and Okano, T. (1994) Temperature modulated solubility-activity alterations for poly(N-isopropylacrylamide)lipase conjugates. J. Biochem. 116, 682-6. (35) Matsukata, M., Aoki, T., Sanui, K., Ogata, N., Kikuchi, A., Sakurai, Y., and Okano, T. (1996) Effect of molecular architecture of poly(N-isopropylacrylamide)-trypsin conjugates on their solution and enzymatic properties. Bioconjugate Chem. 7, 96-101. (36) Ding, Z., Chen, G., and Hoffman, A. S. (1998) Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. J. Biomed. Mater. Res. 39, 498-505. (37) Chilkoti, A., Chen, G., Stayton, P. S., and Hoffman, A. S. (1994) Site-specific conjugation of a temperature-sensitive polymer to a genetically engineered protein. Bioconjugate Chem. 5, 504-7. (38) Stayton, P. S., Shimoboji, T., Long, C., Chilkoti, A., Chen, G., Harris, J. M., and Hoffman, A. S. (1995) Control of protein-ligand recognition using a stimuli-responsive polymer. Nature 378, 472-4. (39) Sano, T., and Cantor, C. R. (1995) Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc. Natl. Acad. Sci. U.S.A. 92, 3180-4.

BC980151F