for One-Pot Affinity - American Chemical Society

EcoRI. INTRODUCTION. Bioaffinity reactions using single- or double-stranded. DNA have been widely used for the detection and purification of biomolecu...
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Bioconjugate Chem. 1998, 9, 719−724

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Water-Soluble Conjugate of Double-Stranded DNA and Poly(N-isopropylacrylamide) for One-Pot Affinity Precipitation Separation of DNA-Binding Proteins Daisuke Umeno, Masafumi Kawasaki, and Mizuo Maeda* Department of Materials Physics and Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Received February 17, 1998; Revised Manuscript Received June 4, 1998

Poly(N-isopropylacrylamide) having a terminal psoralen group was synthesized and covalently bound to plasmid pBR 322 DNA through a photochemical reaction. The resulting conjugate exhibited temperature-responsive precipitation due to the aggregation of poly(N-isopropylacrylamide) chains when heated above 31 °C. This system was used for the one-pot separation of restriction endonuclease EcoRI.

INTRODUCTION

Bioaffinity reactions using single- or double-stranded DNA have been widely used for the detection and purification of biomolecules. Double-stranded (ds) DNA is extensively employed as an affinity ligand (1, 2) for the separation and purification of DNA-binding proteins, such as transcription factors, and immobilized onto solid supports such as sepharose beads (3), synthetic polymer resins (4), and silica gels (5). These approaches, however, have fundamental disadvantages resulting from the insolubilization of DNA ligands. The vibrational and rotational motions of biomolecules immobilized on solid surfaces are restricted, and in most cases, these restrictions reduce the affinity of the ligands for the target molecules as compared with free DNA (6). In addition, solid-liquid heterogeneous reactions are generally slower than solution reactions due to restricted diffusion. Therefore, it is preferable that DNA ligands be immobilized on a water-soluble polymeric supports, enabling the homogeneous binding of the target molecules. However, there must exist a strategy for the collection of the molecules captured by the DNA ligands on the watersoluble carriers. Poly(N-isopropylacrylamide) (polyNIPAAm) is one of the candidates which have both of the water solubility and collectable property: it has a phase transition between water-soluble and -insoluble forms in response to small temperature changes around 31 °C (7) and has recently been successfully utilized for the separation of biomolecules such as antibodies and enzymes (8, 9). In these systems, the binding of ligands and target molecules takes place in homogeneous solution (capturing mode) and the resulting complex is collected upon heatinduced precipitation (collection mode). This strategy is also applicable to the separation of DNA-binding proteins providing there exists a method for the conjugation of DNA with polyNIPAAm. We have studied a method for the synthesis of soluble conjugates of ds DNA and synthetic polymers (10-12) * Author to whom correspondence should be addressed. Phone: +81-92-642-3606. Fax: +81-92-642-3611. E-mail: [email protected].

and recently found that psoralen derivatives are quite suitable for the stable conjugation between them (13). Psoralens are well-known DNA intercalators which crosslink double-stranded DNA when irradiated with a UV light (14). In previous work (15, 16), vinyl groups were introduced into dsDNA in two steps: a vinyl derivative of psoralen was photochemically bound to the ds DNA, then the resultant DNA-macromonomer was copolymerized with acrylamide in water. This resulted in the covalent grafting of polyacrylamide chains to ds DNA. The conjugate between calf thymus DNA and poly(Nisopropylacrylamide) was successfully applied to the separation of DNA-binding dyes (17). In the present study, we synthesized polyNIPAAm having a 4,5′,8trimethylpsoralen (trioxalen) moiety at its terminus. The psoralen-terminated polyNIPAAm was then photochemically bound to DNA. The resulting DNA-polyNIPAAm conjugate was applied to the one-pot affinity separation of restriction endonuclease EcoRI. EXPERIMENTAL PROCEDURES

Materials. N-Isopropylacrylamide (NIPAAm) was obtained from Tokyo Kasei Kogyo and recrystallized from a mixture of benzene and hexane. N,N′-Azobisisobutyronitrile (AIBN) was purchased from Kishida Chemicals. Trioxalen (4,5′,8-trimethylpsoralen) and 3-mercaptopropionic acid (MPA) were obtained from Aldrich. pBR322 DNA was purchased from Fermentas. HindIII and EcoRI were purchased from Boehringer Mannheim. Other reagents and solvents were obtained commercially and used without further purification. Synthesis of Carboxyl-Terminated PolyNIPAAm (PolyNIPAAm-COOH). Semitelechelic polyNIPAAm possesing a carboxyl end group was synthesized by the method described by Takei et al. (18). Briefly, it was prepared by radical polymerization of NIPAAm in dimethylformamide (DMF) using AIBN as initiator and MPA as a chain-transfer agent. The polymer was purified by repeated reprecipitation from DMF-diethyl ether. The molecular weight of the resulting polymer was determined by acid-base titration of the terminal carboxyl group. The phase-transition temperature (cloud point) of aqueous solutions of the polymer was monitored at 500

10.1021/bc980019f CCC: $15.00 © 1998 American Chemical Society Published on Web 08/20/1998

720 Bioconjugate Chem., Vol. 9, No. 6, 1998

Umeno et al.

Scheme 1. Synthetic Route for PsoPNIPAAm

Table 1. Analytical Data for PsoPNIPAAm and Precursors

polyNIPAAm-COOH polyNIPAAm-COOSu PsoPNIPAAm

number-averaged molecular weight

transition temperature (°C)

5400a 6700b 6000c

31.3 30.8

a

Calculated by NaOH titration of the terminal COOH group. Calculated from the UV absorption of the succinimidyl group at 260 nm ( ) 9700). c Calculated by UV absorption of the psoralen moiety at 300 nm ( ) 11 000).

b

nm with temperature raised at the rate of 0.4 °C/min using a spectrophotometer (Hitachi, U-3210). The temperature at which the polymer solution exhibited 10% light transmittance was defined as the cloud point. Activation of the Terminal Carboxyl Group of PolyNIPAAm. The carboxyl group at the polymer terminus was esterified using N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) in a molar ratio of 1.0: 1.2:1.2 in dry DMF at ca. 25 °C for 24 h. The reaction mixture was concentrated in vacuo and poured into diethyl ether to precipitate the product. The activated polymer (polyNIPAAm-COOSu) was purified by repeated reprecipitation from DMF-diethyl ether. Ester formation was confirmed by UV spectrometry (19). Psoralen-Terminated PolyNIPAAm (PsoPNIPAAm). 4′-[[N-(2-Aminoethyl)amino]methyl]-4,5′,8trimethylpsoralen was synthesized according to a method described by Lee et al. (20). The amino derivative of psoralen (0.07 g, 235 µmol) and the activated polyNIPAAm (polyNIPAAm-COOSu; 0.5 g, 94 µmol) were dissolved in dry DMF (total volume, 10 mL). After stirring for 12 h at ca. 25 °C, the solution was concentrated in vacuo and poured into diethyl ether. The precipitate was collected

and washed with 100 mL of diethyl ether. The white powder was dried in vacuo. The polymer thus obtained was dissolved in pure water and its concentration was determined from the UV absorption due to the psoralen moiety (300 nm). Photoimmobilization of PsoPNIPAAm on Plasmid pBR 322 DNA. Plasmid pBR322 DNA was linearized using HindIII and purified by phenol extraction and ethanol precipitation, then resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4). To a solution of linearized pBR 322 (615 µM in base pair, bp) in a micro test tube (Eppendorf, 1.5 mL) was added a TE solution of PsoPNIPAAm. The total volume was then adjusted to 30 µL to give a final DNA concentration of 62 µM bp. The concentration of PsoPNIPAAm was varied from 50 nM to 10 µM (in strand). Each solution was cooled in an ice bath and irradiated for 10 min (ca. 30 mW/cm2) using a 500 W ultrahigh pressure Hg lamp equipped with a high-pass filter (Toshiba, UV31) for 10 min. To the resultant mixture was added 9 µL of aqueous NaOH (1.2 M) in order to make the DNA denatured, and then was combined with 9 µL of gel-loading solution (glycerin:water, 1:1, v/v), quickly loaded to a 1.0% agarose gel, and electrophoresis was performed at 7 V/cm for 1 h in TAE buffer (80 mM Tris-acetate, 1 mM EDTA, pH 7.6). Gel electrophoresis was performed at 10 °C since polyNIPAAm and its conjugates precipitate from aqueous glycerin (25 v/v %) at ambient temperature (12). After electrophoresis, DNA in the gel was stained by ethidium bromide (Figure 1). Thermal Response of the DNA-PolyNIPAAm Conjugate. To a solution of λDNA (48 502 bp) was added PsoPNIPAAm from the stock (100 µM in strand) to give a final concentration of 58 µM bp DNA. The concentration of PsoPNIPAAm was varied from 0 to 50

DNA Conjugate for Affinity Separation

µM (in strand). Samples were irradiated with UV light as described above, and the thermally induced precipitation of the resulting conjugates between λDNA and polyNIPAAm was examined. To the conjugate solution (100 µL) was added 20 µL of aqueous solution of poly(N-isopropylacrylamide) (10 w/v%; 1.7 w/v % in total) and the mixture was then incubated at 40 °C for 3 min and centrifuged (15 000 rpm) at the temperature for 3 min to precipitate the DNA-polyNIPAAm conjugate. After centrifugation, the supernatant was collected and subjected to gel electrophoresis (0.7% agarose, at 7 V/cm for 2 h) in TBE (80 mM Tris-borate, 2.5 mM EDTA, pH 8.0) buffer. After the gel was stained with ethidium bromide, the band due to the conjugate was evaluated using scanning densitometry to estimate the amount of unprecipitated conjugate. Precipitation Separation of EcoRI. DNApolyNIPAAm conjugates were synthesized by the photochemical reaction between pBR 322 (linearized using BsmI, 60 µM bp) and PsoPNIPAAm (20, 40, and 60 µM) in micro test tubes (30 µL in total) as described above. As a reference sample, a mixture of pBR 322 (BsmI digest, 58 µM bp) and PsoPNIPAAm (60 µM), which had not been irradiated with UV light, was also prepared. To these samples were then added EcoRI (60 units) and polyNIPAAm (1.0 w/v %). The solutions (total volume, 60 µL) were incubated at ca. 25 °C for 1 h then heated up to 40 °C in a water bath and centrifuged for 1 min at 40 °C (15 000 rpm). The supernatant (30 µL) was taken from each sample and combined with DNA (pBR 322/ BsmI digest, 0.6 µg). DNA was not added to the supernatant of the reference sample (the mixture of the native DNA and polyNIPAAm), because the DNA in this precedent mixture remained in solution even after the heat and precipitation procedure unless it was conjugated to polyNIPAAm. These solutions were mixed with buffer (total volume, 40 µL; 5.0 mM Tris-HCl, 10 mM NaCl, 1.0 mM MgCl2, 0.1 mM dithioerythritol, pH 7.5), and after incubation for 30 min at 37 °C, samples were subjected to gel electrophoresis (1.0% agarose, at 7 V/cm for 1 h) in TBE buffer (Figure 3). Sequence-Dependent Affinity Separation of EcoRI. DNA conjugates with and without EcoRI binding site were prepared. The former was synthesized by the photoreaction between PsoPNIPAAm (60 µL) and pBR 322 (61 µM bp) which had been linearized using StyI, while the latter was synthesized by the photoreaction between PsoPNIPAAm (60 µM) and pBR 322 (61 µM bp) linearized using EcoRI. A mixture of PsoPNIPAAm (60 µM) and pBR 322 (61 µM bp, linearized using StyI) was also prepared. Each sample was mixed with EcoRI (60 units) and polyNIPAAm (1.0 w/v %) and incubated for 2 h at 4 °C, followed by the heating and precipitation procedure. Thirty microliters of each supernatant was collected, mixed with DNA (0.6 µg of pBR 322 linearized using StyI), and endonuclease reaction was allowed to proceed for 1 h upon the addition of Mg2+ (1 mM). The resultant solution was subjected to gel electrophoresis (1.0 % agarose, at 7 V/cm for 1 h) in TBE buffer (Figure 4). RESULTS AND DISCUSSION

Psoralen-Terminated PolyNIPAAm. The synthesis of psoralen-terminated poly(N-isopropylacrylamide) (PsoPNIPAAm) is shown in Scheme 1. The number-averaged molecular weight of polyNIPAAm was determined by NaOH titration of terminal carboxyl group and the calculated value for the polyNIPAAm-COOH was about

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Figure 1. Gel electrophoresis (1.0 % agarose) of linear pBR 322 (62 µM in bp) which was irradiated with UV light in the presence or absence of PsoPNIPAAm. Samples from lane 2 to 10 were treated with NaOH (0.3 M) before loading. All samples were loaded on a nondenaturing gel. Lanes 1 and 2, control DNA; lane 3, [PsoPNIPAAm] ) 0; lane 4 [PsoPNIPAAm] ) 50 nM; lane 5 [PsoPNIPAAm] ) 100 nM; lane 6 [PsoPNIPAAm] ) 500 nM; lane 7 [PsoPNIPAAm] ) 1 µM; lane 8 [PsoPNIPAAm] ) 5 µM; lane 9 [PsoPNIPAAm] ) 10 µM; lane 10, [PsoPNIPAAm] ) 10 µM, but no UV irradiation.

5400 (ca. 50 mer), which is in good agreement with the previous report (4500) (18). In each step of the synthesis, the nature of end groups was determined and the apparent molecular weights were estimated (Table 1). No significant differences were observed among the three molecular weights. Slight difference should be ascribed to the different methods of evaluation and to the fact that each reaction involved a reprecipitation which could change the molecular weight distribution. PolyNIPAAm is a well known polymer which exhibits a phase transition (7): when an aqueous solution of polyNIPAAm is gradually heated, it abruptly precipitates at 31 °C. The transition temperatures of the polymers prepared in this study were found to be all about 31 °C and the transition between soluble and insoluble form was fully reversible. Photo-Induced Conjugation of PsoPNIPAAm to pBR 322. Psoralens are known to intercalate into DNA double strands in dark and form covalent bonds at their 3,4 and 4′,5′ double bonds with pyrimidines upon nearUV (∼365 nm) irradiation (14). If both sites of the psoralen are reacted, an interstrand DNA cross-linking takes place to make both strands connected to each other. Photoreactivity of the psoralen moiety which had been introduced to the terminus of polyNIPAAm was examined for pBR 322 plasmid DNA according to the literature (21). Figure 1 shows the electrophoretic migration profiles of linearized pBR 322 (EcoRI digest) after the photoreaction with PsoPNIPAAm and further treatment with 0.3 M NaOH. The alkali treatment denatures double-stranded DNA to give single strands, which are characterized by faster electrophoretic migration and the weaker band intensity (lane 2). The alkali denaturation also yielded the single strands from DNA which was irradiated with UV light (lane 3). On the contrary, there was observed significant amount of DNA which ran as ds form even after the alkali-treatment for DNAs which were mixed with PsoPNIPAAm and irradiated with UV light (lanes 4-9). Increasing amount of PsoPNIPAAm fed at the photoreaction resulted in increasing ratio of ds form DNA. When a certain amount of PsoPNIPAAm (1 µM for the 60 µM bp DNA) was used, 100% of DNA behaved as dsDNA (lanes 7-9), while even the highest concentration of PsoPNIPAAm produced no crosslinking in the absence of UV light (lane 10). This is because DNA double strands were cross-linked by the psoralen moiety of PsoPNIPAAm when irradiated with UV light, so that they were not separate to single strands even upon alkali treatment. Therefore, the cross-linked DNA quickly

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Umeno et al. Scheme 2. Affinity Precipitation Separation of DNA-Binding Substances

Figure 2. Gel electrophoresis (0.7 % agarose) of DNApolyNIPAAm conjugates before (a) and after (b) centrifugation at 40 °C. The conjugate of polyNIPAAm and λDNA (58 µM bp) was heated and centrifuged in the presence of 1.7 wt% of NIPAAm homopolymer. Lane 1, DNA alone (control); lane 2, [PsoPNIPAAm] ) 0 µM; lane 3, [PsoPNIPAAm] ) 2 µM; lane 4, [PsoPNIPAAm] ) 5 µM; lane 5, [PsoPNIPAAm] ) 10 µM; lane 6, [PsoPNIPAAm] ) 20 µM.

renatured to the ds form in the buffer (pH 7.6) used for gel electrophoresis and ran as the ds form, while noncross-linked DNA remained single strands and ran with greater mobility. Thus, it was clearly shown that polyNIPAAm was successfully immobilized on the DNA strands with the aid of the terminal psoralen group when irradiated by UV light. There was observed significant retardation in bands corresponding to the ds DNA which was reacted with PsoPNIPAAm, depending on the amount of PsoPNIPAAm used in the photochemical reaction (lanes 5-9). This result should reconfirm the modification of DNA with PsoPNIPAAm, since the conjugation should make the whole molecular size larger. The gradual retardation dependently on the amount of PsoPNIPAAm (lanes 5-9) indicates that one can regulate the degree of modification with polyNIPAAm just by changing the amount of PsoPNIPAAm used in the photoreaction. We also found that the degree of modification can be regulated by changing reaction period, i.e., time period of UV irradiation (data not shown). Temperature-Responsive Precipitation of the Conjugates. In order to take advantage of it for the precipitation separation, the concentration of polyNIPAAm was adjusted to 1.7 wt % by adding NIPAAm homopolymer because we found that a certain concentration of ca. 1.0 wt % is required to ensure reproducible precipitation. The conjugate solution was heated to 40 °C and centrifuged (15 000 rpm) at that temperature to give a white precipitate. The supernatant was carefully collected and subjected to gel electrophoresis. Figure 2 shows the electrophoretic profiles of the conjugate solution (upper lanes) and those of the supernatant after centrifugation at 40 °C (lower lanes). At the upper lanes of 3-6 are observed bands of DNA conjugates with less mobility than the control DNA (lane 1). Similarly to Figure 1, the degree of retardation increased with the amount of

PsoPNIPAAm used in the photoinduced conjugation reaction. After heating and centrifugation, these profiles changed dramatically (lower lanes). The bands due to the DNA conjugate dramatically faded as the amount of polyNIPAAm used in the photochemical reaction was increased. At a certain level (lanes 5 and 6), almost 100% of the conjugate disappeared in the supernatant, due to precipitation. In contrast, the unirradiated mixture of DNA and PsoPNIPAAm did not show any change (lane 2). Samples containing relatively small amounts of PsoPNIPAAm did not show such diminution (lanes 3 and 4). These results indicate that the DNA conjugates acquired thermo-sensitivity derived from the polyNIPAAm chains. We assumed that the polyNIPAAm chains attached to DNA turned to be insoluble above 32 °C and the side chains and polyNIPAAm in bulk condensed via hydrophobic interaction to form a new phase separated from aqueous system, resulting in the distribution (extraction) of DNA conjugated with polyNIPAAm into the newly emerged phase. Affinity Precipitation Separation of EcoRI. The conjugate was applied to the precipitation separation of a DNA-binding protein (Scheme 2). The restriction endonuclease EcoRI recognizes the 5′-GAATTC-3′ unique sequence in pBR 322 (linearized using BsmI; 4363 bp) to give two fragments of 3008 and 1355 bp. The protocol is summarized in Scheme 3. The conjugate between pBR322 and PsoPNIPAAm was added to a buffer solution containing EcoRI. It should be noted that the EcoRI solution was free from magnesium ion in this stage so that DNA scission should not take place (22). It is reported that EcoRI bind tightly (Kd ≈ 1010) to its recognition site even in the absence of magnesium ion (23). The mixture was then centrifuged (15 000 rpm) at 4 °C to give a white precipitate. The supernatant was carefully collected and the endonuclease activity of any remaining EcoRI was evaluated. The results are shown in Figure 3. As seen in lane 1, the supernatant retained its EcoRI activity after the centrifugation (at 40 °C) in the presence of DNA and PsoPNIPAAm (no UV irradiation). In contrast, the EcoRI activity in the supernatant dramatically decreased after centrifugation when the pBR 322-polyNIPAAm conjugate was present (lanes

DNA Conjugate for Affinity Separation

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Scheme 3. Procedure for the precipitation separation of EcoRI and for the evaluation of precipitation/separation efficiencya

a DNA-polyNIPAAm conjugates were added to EcoRI (60 units) and the concentration of polyNIPAAm was adjusted to 1 wt %. The mixtures were heated at 40 °C and centrifuged to yield a white precipitate. The amount of EcoRI remaining in the supernatant was evaluated by its endonuclease activity.

2-4). The conjugate thus seems to be responsible for the decrease in the amount of EcoRI in the supernatant. These decrease in activity is proportional to the concentration of PsoPNIPAAm used in the modification of the DNA, reflecting the difference in the efficiency of precipitation of the conjugates. These results strongly corroborate the affinity separation system illustrated in Scheme 2. To ascertain that the DNA conjugate functions as a sequence-dependent affinity separator for EcoRI, two types of conjugates were prepared and used for the precipitation separation of EcoRI: a conjugate between PsoPNIPAAm and pBR 322 digested with StyI and a conjugate between PsoPNIPAAm and pBR 322 digested with EcoRI. The conjugates differ only in that the former has an EcoRI-binding site on its strand, while the latter does not. An aqueous solution of restriction endonuclease EcoRI (60 units), conjugate, and NIPAAm homopolymer (1.0 w/v % in total) were incubated, and then the mixture was heated to 40 °C and centrifuged at that temperature. The supernatant was collected and its EcoRI activity was determined. The result is shown in Figure 4. Supernatant retained a certain level of EcoRI activity to give two fragments (1371 and 2992 bp) when the DNA-polyNIPAAm conjugate without the EcoRI-binding site was used as a separation agent (lane 4). The enzymatic activity was almost the same as that for the simple mixture of DNA and PsoPNIPAAm (no UV irradiation, lane 2) in the supernatant after the heating and precipitation. This indicates that the DNA-polyNIPAAm conjugate without an EcoRI binding site failed to complex with EcoRI. On the other hand, there was no sign of EcoRI activity in the supernatant when the DNA-polyNIPAAm conjugate having an EcoRI site was used (lane 3). These results indicate that ds DNA bearing a recognition site for EcoRI acts as an affinity ligand for EcoRI and that the presence of this site is exclusively responsible for the separation of EcoRI. It has been clearly demonstrated that EcoRI was captured by the conjugate bearing its recognition site and

Figure 3. Precipitation separation of EcoRI using DNApolyNIPAAm conjugates. EcoRI solution (60 units) and DNApolyNIPAAm conjugates were mixed and the concentration of polyNIPAAm was adjusted to 1.0 w/v %. The mixtures were then heated at 40 °C and centrifuged. To a fixed volume of the supernatant, substrate DNA (pBR 322/BsmI digest) and the reaction buffer were added, followed by incubation for 30 min at 37 °C. The solution was then subjected to gel electrophoresis. Lane 1, mixture of DNA (61 µM) and PsoPNIPAAm (60 µM); lanes 2-4, conjugates between DNA (61 µM) and polyNIPAAm (lane 2, 20 µM; lane 3, 40 µM; lane 4, 60 µM). Lane 5 is a control (BsmI digest of pBR 322).

precipitated from aqueous solution as illustrated in Scheme 2. We have observed endonuclease activity in the precipitated fractions, but the quantitative evaluation of the recovery was not performed. The separation capacity of this system also remains to be clarified. Such information could be obtained if this system were used on a larger scale and could be much refined if the DNA sequence of the conjugate were properly designed. At present, plasmid DNA with a tandem array of EcoRI sites is being developed for further investigations of this separation system. There remains an important question before applying this system to the practical uses: how could we recover the proteins isolated by this system? We assumed that it would be possible if the precipitation fraction was dissolved in fresh water and a certain concentration of salt was added. According to the literature (23), the interaction betwen EcoRI and its recognition sequence becomes drastically weak by increasing NaCl concentration, and EcoRI bound to the sepharose column carrying its recognition sequence could be easily eluted by adding ca. 0.3 M of KCl (24) or by adding 1.5 M of NaCl (25) to mobile phase. On the other hand, the transition temperature of polyNIPAAm is known to decrease with increasing salt concentration (7). According to our observation, it becomes as low as 15 °C at the NaCl concentration of 1.5 M and becomes insoluble at room temperature. Taking advantages of these two facts, EcoRI collected by our system would be separated from DNA-polyNIPPAm conjugate upon addition of NaCl (1.5 M) and isolated as supernatant fraction at room temperature. At present, however, we have no data supporting this conjecture. We have demonstrated the affinity precipitation separation of DNA-binding molecules using a DNA-polyNIPAAm conjugate. This conjugate may provide a rapid and widely applicable separation technique, since the complexation with the target takes place under homogeneous conditions and the separation is accomplished

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Figure 4. Sequence-dependent affinity separation of EcoRI. EcoRI (60 units), polyNIPAAm (1.0 w/v %), and conjugates were mixed and incubated for 2 h at 4 °C, then subjected to the heat and precipitation procedure. Supernatant was collected from each samples and the enzymatic activity of any residual EcoRI was evaluated. To a substrate DNA (0.6 µg of pBR 322 linearized using StyI) was added 30 µL of the supernatant, and the endonuclease reaction was performed for an hour upon adding Mg2+ (1.0 mM). The resultant solution was subjected to gel electrophoresis. Lanes 1 and 5, control DNA (pBR 322); Lane 2, the mixture of pBR 322 (62 µM) digested using Sty I and PsoPNIPAAm (60 µM); lane 3, the conjugate between PsoPNIPAAm (60 µΜ) and pBR 322 digested using StyI; lane 4, conjugate between PsoPNIPAAm (60 µM) and pBR 322 digested using EcoRI.

in a single reaction vessel. The present method allows us to use double-stranded DNA as an affinity ligand, so that one may collect any ions, molecules, or macromolecules which bind to the DNA. ACKNOWLEDGMENT

We thank Dr. Scott J. McNiven (Research Center for Advanced Science and Technology, the University of Tokyo) for helpful discussion. This work was supported in part by a Grant-in-Aid for Scientific Research from Ministry of Education, Science, Sports and Culture of Japan. Financial support by the General Sekiyu Research and Development Encouragement and Assistance Foundation is also acknowledged. LITERATURE CITED (1) Kadonaga, J. T. (1991) Purification of Sequence-Specific Binding Proteins by DNA Affinity Chromatography. Methods Enzymol. 208, 10-23. (2) Jarrett, H. W. (1993) Affinity Chromatography with Nucleic Acid Polymers. J. Chromatogr. 618, 315-339. (3) Kadonaga, J. T., and Tjian, R. (1986) Affinity Purification of Sequence-Specific DNA Binding Proteins. Proc. Natl. Acad. Sci. U.S.A. 83, 5889-5893. (4) Kawaguchi, H., Asai, A., Ohtsuka, Y., Watanabe, H., Wada, T., and Handa, H. (1989) Purification of DNA-Binding Transcription Factors by Their Selective Adsorption on the Affinity Latex Particles. Nucleic Acids Res. 17, 6229-6240. (5) Goss, T. A., Bard, M., and Jarrett, H. W. (1991) HighPerformance Affinity Chromatography of Messenger RNA. J. Chromatogr. 588, 157-164. (6) Bunemann, H. (1982) Immobilization of Denatured DNA to Macroporous Supports: II. Steric and Kinetic Parameter of Heterogeneous Hybridization Reactions. Nucleic Acids Res. 10, 7181-7196. (7) Schild, H. G. (1992) Poly(N-isopropylacrylamide): Experiment, Theory and Application. Prog. Polym. Sci. 17, 163249. (8) Chen, J. P., Yang, H. J., and Hoffman, A. S. (1990) PolymerProtein Conjugates. II. Affinity Precipitation Separation of

Umeno et al. Human Immunogammaglobulin by a Poly(N-isopropylacrylamide)-Protein A Conjugate. Biomaterials 11, 631-634. (9) Takei, Y. G., Matsukata, M., Aoki, T., Sanui, K., Ogata, N., Kikuchi, A., Sakurai, Y., and Okano, T. (1994) TemperatureResponsive Bioconjugates. 3. Antibody-Poly(N-isopropylacrylamide) Conjugates for Temperature-Modulated Precipitations and Affinity Bioseparations. Bioconjugate Chem. 5, 577-582. (10) Maeda, M., Hirai, A., and Takagi, M. (1989) Gel Electrophoretic Demonstration of Pseudo-Grafting of Polyacrylamide onto λ Phage DNA. Chem. Lett. 1989, 1831-1834. (11) Maeda, M., Hirai, A., and Takagi, M. (1991) PseudoGrafting of Polyacrylamide onto HindIII Restriction Fragments of λ DNA. Reactive Polym. 15, 103-109. (12) Maeda, M., Nishimura, C., Inenaga, A., and Takagi, M. (1993) Modification of DNA with Poly(N-isopropylacrylamide) for Thermally Induced Affinity Separation. Reactive Polym. 21, 27-35. (13) Kashiwagi, S., Ohmori, K., Maeda, M., and Takagi, M. (1992) Efficient Coupling of DNA on a Silica Support with Psoralen Derivatives as Anchoring Agents for High-Performance Liquid Affinity Chromatography. Anal. Sci. 8, 261263. (14) Cimino, D. G., Gamper, H. B., Isaacs, S. T., and Hearst, J. E. (1985) Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry, Ann. Rev. Biochem. 54, 1151-1193. (15) Maeda, M., Nishimura, C., Umeno, D., and Takagi, M. (1994) Psoralen-Containing Vinyl Monomer for Conjugation of Double-Helical DNA with Vinyl Polymers. Bioconjugate Chem. 5, 527-531. (16) Maeda, M., Umeno, D., Nishimura, C., and Takagi, M. (1996) Conjugation of DNA with Functional Vinyl Polymers by Using Vinyl Derivative of Psoralen as a Linker. In Hydrogels and Biodegradable Polymers for Bioapplications (R. M. Ottenbrite, S. J. Huang, and K. Park, Eds.) pp 197208. American Chemical Society, Washington, DC. (17) Umeno, D. and Maeda, M. (1997) Poly(N-isopropylacrylamide) Carrying Double-Stranded DNA for Affinity Separation of Genotoxins. Anal. Sci. 13, 553-556. (18) Takei, Y. G., Aoki, T., Sanui, K., Ogata, N., Okano, T., and Sakurai, Y. (1993) Temperature-Responsive Bioconjugates. 1. Synthesis of Temperature-Responsive Oligomers with Reactive End Groups and Their Coupling to Biomolecules. Bioconjugate Chem. 4, 42-46. (19) Miron, T. and Wilchek, M. (1982) A Spectrophotometric Assay for Soluble and Immobilized N-Hydroxysuccinimide Esters. Anal. Biochem. 126, 433-435. (20) Lee, B. L., Murakami, A., Blakes, K. R., Lin, S.-B., and Miller, P. S. (1988) Interaction of Psoralen-Derivatized Oligodeoxyribonucleoside Methylphosphonates with SingleStranded DNA. Biochemistry 27, 3197-3203. (21) Saffran, W. A., Welsh, J. T., Knobler, R. M., Gasparro, F. P., Cantor, C. R., and Edelson, R. L. (1988) Preparation and Characterization of Biotinylated Psoralen. Nucleic Acids Res. 16, 7221-7231. (22) Halford, S. E. and Johnson, N. P. (1980) The EcoRI Restriction Endonuclease with Bacteriophage Lambda DNA. Equilibrium Binding Studies. Biochem. J. 191, 593-604. (23) Terry, B. J., Jack, W. E., Rubin, R. A., and Modrich, P. (1983) Thermodynamic Parameters Governing Interaction of EcoRI Endonuclease with Specific and Nonspecific DNA Sequences. J. Biol. Chem. 258, 9820-9825. (24) Vlatakis, G., and Bouritos, V. (1991) Sequence-Specific DNA Affinity Chromatography: Application to the Purification of EcoRI and SphI. Anal. Biochem. 195, 352-357. (25) Blanks, R. and McLaughlin, W. (1991) An Oligonucleotide Affinity Column for the Isolation of Sequence-Specific DNA Binding Proteins. Nucleic Acids Res. 16, 10283-10299.

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