Specific base recognition of oligodeoxynucleotides by capillary affinity

Tomohiro Sawa and Mitsuru Akashi*. Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University,. Korimoto ...
0 downloads 0 Views 668KB Size
Download PDF
1920

Anal. Chem. 1992, 64, 1920-1925

Specific Base Recognition of Oligodeoxynucleotides by Capillary Affinity Gel Electrophoresis Using Polyacrylamide-Poly(9-vinyladenine) Conjugated Gel Yoshinobu Baba' and Mitsutomo Tsuhako Kobe Women's College of Pharmacy, Kitamachi, Motoyama, Higashinada-ku, Kobe 658, Japan

Tomohiro Sawa and Mitsuru Akashi* Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, Korimoto, Kagoshima 890, Japan

Eiji Yashima Department of Applied Chemistry, Faculty of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan

Poly(9-vinyladenhe) was synthesized and utlked as an afflnlty macrollgand entrapped wlthln the gel matrlx. Base-speclflc separation of ollgodeoxynucleotldes was achleved wlth hlgh resolution and hlgh speed by electrophorerls, ushrgcapillaries fllkd wlth conJugatedpolyacrylamld~ly(9-vlnyl~enlne) gel. Ollgothymklyllc acids were selectlvety separated from the mlxture of ollgothymldyllc and ollgodmcyadenyllc acids by utlllzlng a s p e c k hydrogen bondlng between poly(9-VInyladenlne) and ollgothymldyllc aclds. Mlgratlon tlme and resolullonof ollgodeoxynucleotldeswere Influencedby several parameters, such as the dze of poly(9-vlnyladenlne), caplllary temperature, and concentratlonoof poly(9-vlnyladenlne) and urea. Some guldellms are presented, bared on the theoretkal fonnulatlon of the effect of these parameters, In order to flnd optlmum electrophoretk condttknr. Analytkal capMary affhlty gel electrophorerls was developed for the selectlve and senrltlve bare recognltlon of ollgodeoxynucleotldes wlth efflclencks as hlgh as several lod plates/m by udng a ureagel caplllary wlth poly(9-vlnyladenlne) and temperatureprogrammlng.

INTRODUCTION High-resolution separations of DNA and RNA have been widely achieved using gel electrophoresis1and HPLC.2 More recently, capillary gel electrophoresis (CGE) has been developing rapidly. The resolving power of CGE3-l1 is much higher than those of gel electrophoresis5*6 and HPLC.lO

* To whom all correspondence should be addressed.

(1)Rickwood, D., Hames, B. D., Eds. Gel Electrophoreis of Nucleic Acids-A Practical Approach, 2nd ed.; IRL Press: Oxford, U.K., 1990. (2)KrstuloviC, A. M., Ed. CRC Handbook of Chromatography, Nucleic Acids and Related Compounds; CRC Press: Boca Raton, FL, 1987; Vol. 1, Parts A and B. (3)Cohen, A. S.;Najarian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988,85,9660-9663. (4)Guttman, A.;Cohen, A. S.;Heiger, D. N.; Karger, B. L. Anal. Chem. 1990.62.137-141. (5) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; DCunha, J.; Smith, L. M. Anal. Chem. 1990,62,-903. (6)Swerdlow, H.; Wu, S.; Harke, H.; Dovichi, N. J. J. Chromatogr. 1990.516.61-67. -- (7) Paulus, A.; Ohms, J. I. J. Chromatogr. 1990,507,113-123. (8) Yin,H.-F.;Lux, J. A.; Schomburg,G. J.HighResolut. Chromatogr. 1990,13,624-627. (9)Baba, Y.; Matsuura, T.; Wakamoto, K.; Tsuhako, M. Chem. Lett. 1991,371-374.

.

~

0003-2700/92/0364-1920$03.00/0

Although affinity gel electrophoresis12and high-performance affinity chromatography (HPAC),13which are alternative modes of gel electrophoresis and HPLC, including a biospecific affinity ligand, are both becoming important tools for biospecific separation of DNA and RNA, affinity gel electrophoresis has entered the CGE field more slowly. Development of an affinity mode of CGE, namely, capillary affinity gel electrophoresis (CAGE), will open up new horizons for the biospecific separation of DNA and RNA with ultrahigh resolution as well as characterizing molecular properties of DNA and RNA by analyzing biospecific interactions. Biospecific separation in affinity gel electrophoresisis based on a change in electrophoretic mobility of the analyte caused by some biospecific interaction between the analyte and the affinity ligands. The methods of incorporation of affinity ligands into the gel matrix are classified into three groups:14 (1)solubilized ligand in the gel matrix (Figure lA), macroligand entrapped within the gel matrix (Figure lB), and immobilized ligand chemically bonded to the gel matrix (Figure 1C). Guttman and Cooke15recently reported CAGE using capillaries filled with linear polyacrylamide, in which ethidium bromide was solubilized as a ligand, for specific separation of double-stranded DNA fragments by the intercalation of ethidium cations. However, specific base recognition of nucleic acids has never been realized by using CAGE. To design such a CAGE system, new gels incorporating a base-recognizable ligand should be developed. Few novel ligands have been introduced for conventional affinity gel electrophoresis, e.g., poly(vinylnucleobase)~~J~ and phenylboronate polyacrylamide gels.'* In this paper, we present the CAGE principle in specific base recognition of nucleic acids and ita application. We synthesized poly(9-vinyladenine) (PVAd) as a macroligand and produced capillaries filled with cross-linked polyacrylamide gel conjugated with PVAd. The conjugated gel-filled (10)Baba, Y.; Matsuura, T.; Wakamoto, K.; Tsuhako, M. J. Chromatogr. 1991,558,273-284. (11)Baba, Y.; Matsuura, T.; Wakamoto, K.; Morita, Y.; Nishitsu, Y.; Tsuhako, M. Anal. Chem. 1992,64,1221-1225. (12)Takeo, K.Adu. Electrophoresis 1987,1, 229-279. (13)Fassina, G.; Chaiken, I. M. Adu. Chromatogr. 1987,27,247-297. (14)Horejsi, V.;Ticha, M. J. Chromatogr. 1986, 376,49-67. (15)Guttman, A.; Cooke, N. Anal. Chem. 1991,63,2038-2042. (16)Pitha, J. Anal. Biochem. 1975,65,422-426. (17)Yashima, E.;Suehiro, N.; Akashi, M.; Miyauchi, N. Chem. Lett. 1990,1113-1116. (18)Igloi, G.L.; Kossel, H. Nucleic Acid Res. 1986,19, 68816898. 0 1992 American Chemical Society

ANAL.YTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

Flgurr 1. Schematlc representations of afflnky ligands in polyacrylamlde gel: (A) mobile ligand method: (B) macroilgand method: (C) immoblllzed llgand method.

9-(2'-chlorwthyl)adenine

9-vinyladenine

poly(9-vinyladenine)

Flgure 2. Scheme of synthetic route for poly(9-vlnyladenlne). capillary performed a base-specific separation of a mixture of oligodeoxyadenylic acids [oligo(dA)l and oligothymidylic acids [oligo(dT)I. The specificity was achieved on the basis of the decrease in electrophoreticmobility of oligo(dT), which formed complementary hydrogen bonding with PVAd, whereas the mobility of oligo(dA) was unchanged even in the presence of PVAd, due to no interaction with PVAd. Electrophoretic mobility of oligo(dT) was highly sensitive to the size of PVAd, capillary temperature, and the concentration of PVAd and urea. The capillary temperature-programming technique was effective for the high-performance base-specific separation of a mixture of oligodeoxynucleotides. This is the first report on the specific base recognition of oligodeoxynucleotides using CAGE.

EXPERIMENTAL SECTION Materials. Tris(hydroxymethy1)aminomethane (Tris),boric acid, and urea were of reagent grade from Wako (Osaka, Japan). Acrylamide,NJV'-ethylenebis(acry1amide)(BIS),NJV,"JV'-tetramethylethylenediamine (TEMED), and ammonium peroxodisulfate were of electrophoretic grade from Wako. Oligodeoxyadenylicacids, (dA)lz-ls,and oligothymidylicacids, (dT)12-1st were purchased from Pharmacia (Uppsala, Sweden). (dT)ls was chemically synthesized using an Applied Biosystems Inc. (ABI, Foster City, CA) Model 391 DNA synthesizer. Samples were diluted to 2.5 unita/500 pL with distilled water and stored at -18 OC until use. Poly(9-vinyladenine) (PVAd) was prepared according to the literature'9~2~ with a slight modification as follows (Figure 2). The 9-(2'-chloroethyl)adenine was prepared in a similar manner to that reported.lg To 9-(2'-chloroethy1)adenine (1.98 g) in dry acetonitrile (200mL) was added l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.82 g) at room temperature. After the reaction mixture was refluxed with stirring for 60 h, acetonitrile was removed under reduced pressure. The product, 9vinyladenine,was recrystallized from a benzene/ethanol mixture (9:l v/v); yield, 1.09 g (68%). The 9-vinyladenine was polymerized in H20 in a sealed tube in vacuo after repeated degassing. Ammonium peroxodisulfate was used as a radical initiator. After 48 h, the reaction mixture was poured into an excess of methanol and the polymer was collected by filtration and then dried in vacuo at 60 OC. PVAd thus obtained was fractionated using ultrafiltration (Amicon standard cell 8200) with an Amicon membrane (molecularweight cutoff: 10 OOO, 30 000, and 50 OOO) under nitrogen pressure (2.0 kg/cm2). Three PVAd samples having molecular weights ranging from 10 OOO to 30 000, from 30 OOO to 50 OOO, and less than 10 000 were obtained. Apparatus. Capillary gel electrophoretic separations were carried out by using an AB1 Model 270A capillary electrophore(19) Akashi, M.; Iwasaki, H.;Miyauchi, N.; Sato, T.; Sunamoto, J.; Takemoto, K. J. Bioact. Compat. Polym. 1989,4, 124-136. (20) (a) Yashima, E.;Tajima, T.; Miyauchi, N.; Akashi, M. BiopolyHayashi, M.; mers, in press. (b)Akashi, M.; Yamaguchi, M.; Miyata, H.; Yashima, E.; Miyauchi, N. Chem. Lett. 1988, 1093-1096. (c) Yashima, E.;Shiiba, T.; Sawa, T.; Miyauchi, N.; Akaahi, M. J . Chrornatogr. 1992, 603,111-119.

1921

sis system. The electropherograms were processed on a Hitachi (Tokyo,Japan) D-2500integrator. Polyimide-coatedfused-silica capillaries (375-rm 0.d. and 100-rm i.d., GL Sciences, Tokyo, Japan) were used, with an effective length of 22 cm and a total length of 42 cm. The temperature of the agitated air surrounding the capillary was maintained at a constant temperature within h0.1 "C. Oligonucleotides were detected at 260 nm. Procedure. The buffer was a mixture of 0.1 M Tris and 0.1 M boric acid with an appropriate concentration of urea (pH 8.6) for the preparation of the gel-filled capillaries, as well as the running buffer. To provide an optical detection window,0.5cm of polyimide coating was burned off at approximately 20 cm from the outlet end. The char was removed with methanol. Capillaries filled with polyacrylamide gel (8%T and 5 % C) were prepared by the injection of degassed polymerizingsolution into uncoated capillaries and in situ polymeri~ation.~J~ A similar method was used to produce capillaries filled with polyacrylamide-PVAd conjugated gel (8%T, 5% C, and 0.02-0.1% PVAd) by using a polymerizing solution consisting of PVAd, acrylamide, and BIS. The percentage of PVAd was calculated by the equation, 100 [PVAd(g)l/{[acrylamide(g)l + [BIS(g)l+ [PVAd(g)]}. Gel-filled capillarieswere mounted in the AB1Model 270A system and run with buffer solution at 9 kV (214V/cm). Samples were electrophoretically injected into the capillary by applying a voltage of 5 kV for 0.1-1 s.

RESULTS AND DISCUSSION Principle of Specific Base Recognition of Oligodeoxynucleotides. Affinity gel electrophoresis is a technique useful for the separation and the characterization of biopolymers utilizing biospecific interaction between biopolymer and an affinity ligand in the medium. As shown in Figure 1,an affinity ligand may be either soluble or immobilized in the gel matrix. In order to realize specific base recognition using affinity gel electrophoresis, the macroligand or the immobilized ligand including bases complementary to the oligonucleotide sample solute should be used, since such ligands would bind tightly to oligonucleotides by specific interactions, Le., base-paired complexes were formed by the complementary hydrogen bonding and stabilized by the stacking interaction of bases.21 On the other hand, a mobile monomer ligand interacts very weakly with oligonucleotide. Natural oligonucleotide, which is used as an affinity ligand for highperformance affinity chromatography,22 is irrelevant for the ligand of affinity gel electrophoresis,because anionic ligands result in unavoidable local electroosmotic flow, which diminishes the high resolving power of capillary affinity gel electrophoresis. Synthetic analogues of nucleic acids, therefore, will be valuable as affinity ligands in capillary affinity gel electrophoresis. We synthesized poly(9-vinyladenine) (PVAd) as a watersoluble polynucleotide analogue having a polyvinyl instead of the sugar-phosphate backbone in natural polynucleotides, as shown in Figure 2, and used it as an affinity macroligand. PVAd possesses some advantages in avoiding electroosmotic flow and is stable against chemical and enzymatic hydrolysis over natural polynucleotides. Additionally, PVAd forms complexes in vitro with the complementary strand of natural polynucleotides, such as poly(U), by hydrogen bonding established by imino proton NMR, CD, and UV spectroscopies.208 It is an excellent affinity ligand for the basespecific separation of oligonucleotides in HPLC using PVAdimmobilized silica gel.2ob-c In the present method, gel-filled capillaries for affinityelectrophoresis were prepared by in situ polymerization from a solution of acrylamide, BIS,and PVAd in the capillary. If a sufficiently large macromolecule is added to the solution of (21) Akashi, M.;Takemoto, K. Ado. Polym. Sci. 1990, 97,107-146. (22) Goss, T.A.; Bard, M.; Jarrett, H. W. J. Chrornatogr. 1990,508,

219-287.

1022

ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

Flgurr 9. Separatlon of ollgodeoxynucleotldesby using (A) capillary gel electrophoresis and (B) caplllary affinity gel electrophoresis.

acrylamide monomers, then, after the copolymerization reaction, a gel is formed in which the macromolecule is entrapped and thus effectively immobilized. The efficiency of immobilization depends on the ratio of macromolecular size and gel porosity. PVAd was effectively immobilized into the polyacrylamide gel system, since it showed no electrophoretic mobility in slab gel electrophoresis. We chose (dA)lz-ls and (dT)lz-18as model substrates to demonstrate the base-specific recognition of oligo(dT) by PVAd. Each component with the same chain length should exhibit almost the same electrophoretic mobility in capillary gel electrophoresis without affinity media (Figure 3A). On the other hand, the mobility of oligo(dT) will show strong retardation with PVAd (Figure 3B) despite no change in mobility of oligo(dA). According to the mechanism shown in Figure 3, specific recognition of d T over dA was achieved using capillary affinity gel electrophoresis. Theoretical aspects of affinity gel electrophoresishave been established.12J4,2+% The interaction of oligcdeoxynucleotides (N) and PVAd (L) is expressed as follows:

where N.L is the complex, square brackets represent the concentration, and K, is the apparent association constant. The electrophoretic mobility and migration time of N are expressed as po and to in the absence of affinity ligand and p and t in the presence of affinity ligand, respectively. Although the value of [Ll, which is the free ligand concentration in the gel, depends on the concentration of N, the free ligand concentration, [Ll, is practically equal to the total L concentration in the gel, [LI,, when [LI, is much higher than the total N concentration, [NI,. Equation 2, therefore, becomes

t = t,(l + K,[Ll,) (3) This equation predicts that an increase in [L], and K, will lead to an increase in migration time. Separation of (dA)lz-leand (dT)12-18Using Capillary Gel Electrophoresis with and without Affinity Ligand. We first measured the migration time, t o in eq 3, of (dA),z-ls and (dT)lz-la using polyacrylamide gel (8% T and 5% C) filled capillaries without affinity microligand to examine the effect of base composition on the electrophoretic mobility. A relatively high gel concentration was selected for the separation of oligodeoxynucleotides, because the high % T gel filled capillary gave better resolution of smaller oligodeoxynucleotides. Figure 4 demonstrates that all components consisting of (23)Takeo, K.; Nakamura, S. Arch. Biochem. Biophys. 1972,153,l-7. (24)Dunn, B.M.;Chaiken, I. M. h o c . Natl. Acad. Sci. U.S.A. 1974, 71,2382-2385. (25)Horejsi, V.;Ticha, M.; Kocourek, J. Biochim. Biophys.Acta 1977, 499,290-300. (26)Dunn, B.M.;Chaiken, I. M. Biochemistry 1975,14,2343-2349.

10

15

min

Flgurr 4. Separation of (dA)lp-18and (dT)lz-18by caplllary polyacrylamlde gel electrophoresis. Sample: (dA)12-1S (A); (dT)lp-18 (B); mixture of (dA)12-18and (dT)lp-18(C). Conditions: caplllary 100-pm l.d., 375pm o.d., 42-cm length, 22-cm effectlve length; runnlng buffer 0.1 M Trls-borate and 7 M urea, pH 8.6; gel, 8% T and 5 % C; caplllay temperature, 30 'C; fleld, 214 V/cm; current, 9 pA; Injection, 5 kV for 1 s; detection, 260 nm.

(dA)12-18 (Figure 4A) or (dT)lz-le (Figure 4B) are baseline resolved into seven bands, respectively, within 15min. The migration time of each pair of oligodeoxynucleotides with the same chain length differs slightly, e.g., 12.54 min for ( d A h and 13.20 min for (dT)lz. Consequently, 14 oligodeoxynucleotides in a mixture of (dA)tz-18and (dT)12-18were separated completely within 15 min, as shown in Figure 4C. Plate number was achieved to be (1-8) X 106 plates/m. The effect of base composition on mobility is understood in conventional278 and capillary27b gel electrophoreeis and is expressed as a simple equation.27 Homooligomers of the same chain lengths exhibited some differences in mobility and the order of mobility was C > A > T > G with the mobility relationship between the homologous series being C10.e = AIO= T8.5 = G6.9.z7a The mobility relationship (A10 = T9.5) obtained from Figure 4 was roughly in agreement with that reported in the literature. The electropherogram in the presence of PVAd in Figure 5A is entirely different from that in Figure 4. Some oligo(dT), 12mer (14.84 min) and 13mer (17.24 min), electrophoresced very slowly, caused by the interaction with PVAd even in the presence of excess denaturing agent, Le., 7 M urea. Some band-broadening of these peaks also occurred in comparison with Figure 4B. Bands corresponding to larger oligo(dT), e.g., 15-18mer, were broadened considerably, presumably due to strong binding to PVAd. On the other hand, the migration time of each component of (dA)12-18was constant in comparison with Figures 4 and 5 within batchto-batch error (3% ) in the production of gel-fiiedcapillaries.11 The results demonstrate that the capillary affinity gel electrophoresis developed here recognizes the base composition of oligodeoxynucleotideswith high specificity and sensitivity. Factors Affecting Specific Base Recognition of Oligodeoxynucleotides. We first examined the effect of the polymerization degree of PVAd on the electrophoretic behavior of oligodeoxynucleotides. Three PVAd prepared by the fractionation of crude polymerized product have a molecular weight range of less than 10 000 (PVAd-l), 10 rn (27)(a) Frank, R.;Koster, H. Nucleic Acids. Res. 1979,6,2069-2087. (b) Guttman, A.;Nelson, R. J.; Cooke, N. J.Chromatogr. 1992,593,297303.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

II! 1

50

(C)

l,l/~ 10

"C

30 "C (0-5 min) 50 "C (5-20 min)

15 min

0

Flguo6, Capillary afflnity gel electrophoresisof (dA)lz-la and (~T)Iz-I~. The gd COntalned 8% T, 5 % C, and 0.05 % poly(9-vinyladenine) with a molecular weight range of 10 000-30 000. Capiilay temperature: 30 OC (A): 50 OC (B); 30 O C for 0-5 min and 50 O C for 5-20 min (C). Other conditions are as in Figure 4. Table I. Migration Time (rnin) of Oligodeoxynucleotides in Capillary Affinity Gel Electrophoresis. Using Poly(9-vinyladenine) (PVAd) with Different Molecular Weights

sample

without PVAd

PVAd-l*

PVAd-2c

PVAd-3d

(dT)iz (dT)ia (dT)16 (dA112

13.20 14.01 14.35 12.54 13.34 13.62

13.50 14.56 14.84 12.81 13.72 14.00

14.26 15.62 17.62 12.62 13.40 13.66

13.76 16.00 20.81 12.55 13.31 13.58

(dA)l5 (&)le

1929

Conditions: 100-pm i.d., 375-pm o.d., 42-cm length, 22-cm effective length; gel contained 8% T and 5% C with or without 0.05 % PVAd; running buffer 0.1 M Tris, 0.1 M boric acid, and 7 M urea, pH 8.6; field, 214 V/cm; current, 10 pA. Molecular weight range of PVAd is less than 10 OOO. Molecular weight range of PVAd is 10 00030 OOO. d Molecular weight range of PVAd is 30 000-50 OOO. 30 OOO (PVAd-2),and 30 000-50 OOO (PVAd-3). The number of adenine bases including these PVAd are estimated to be lees than60 (PVAd-l),60-180 (PVAd-2),and 180-310 (PVAd3), respectively. Conjugated gel-filled capillaries were produced using these PVAd (0.05 % ) and used to measure the migration time of oligodeoxynucleotides. Table I compiles migration times thus obtained and those measured without PVAd for comparison. The number of bases in PVAd is directly related to the apparent association constant, K,;i.e., an increase in the number of bases induces an increase in K,, which results in the migration time increasing as predicted from eq 3. The results in Table I illustrate that the migration times of (dT)15and (dT)16increase with the number of bases on PVAd, but that those of (dT)12 and oligo(dA),are insensitive to the change in the number of bases on PVAd. These results suggest that these PVAd are suitable for the base-specific separation of the mixture of oligo(dT) and oligo(dA) containing more than 15 bases and larger PVAd will be required for the base recognitionof smaller oligonucleotides such as (dT)12. Of these, PVAd-2 was sufficient for use in base-specific separation without an appreciable decrease in resolving power. On the other hand, PVAd-1 and -3 were inadequate for base recognition, because PVAd-1had very low base specificity due to a weak interacting ability with oligo(dT) and CAGE using PVAd-3 exhibited poorer resolution due to severe band-broadening caused by too strong interaction. We have therefore used PVAd-2 to

0.05

Concentration of poly(9-vinyladenine)

0.1

("0)

Figure 6. Effect of the concentration of poly(-vinyladenine) on the migration time of oiigodeoxynucieotldes. Conditions are as in Figure 5 except for the concentration of poly(9-vinyladenine).

test other separation conditions for capillary affinity gel electrophoresis. We then examined the effect of the PVAd concentration ([LIJ on the base-recognizingseparation of a mixture of oligo(dT) and oligo(dA). Figure 6 shows that an increase in [L], leads to an increase in migration time of oligo(dT),as expected from eq 3, but does not affect that of oligo(dA). Although eq 3 predicted the linear relationship between the migration time of oligo(dT) and [Lit, the resultant lines in Figure 6 were slightly curved. This might be caused by the formation of other complexes, e.g., N2.L, in addition to the N.L complex formation which was only assumed in the theory. Small oligo(dT) such as (dT)12,which showed very weak interaction with all PVAd at 0.05%, as in Table I, exhibited detectable interaction with PVAd at 0.1% PVAd-2. However, a (dT)ls band was not observed at 0.1% PVAd, due to broadening. We concluded from the results that an appropriate concentration of PVAd should be selected according to the number of bases of the oligo(dT) for the effective base-specific separations. Figure 7 illustrates that an increase in the concentration of urea results in a decrease in migration time of oligo(dT) in contrast to the effect of [L], in Figure 6. Migration times of oligo(dA) were also insensitive to a change in the concentration of urea as well as [L],. Urea has been extensively used in the gel electrophoresis as a denaturant to abolish the secondary structure of DNA and RNA.' We first thought that base specificity of PVAd realized by the formation of hydrogen bonds with oligo(dT) would disappear in the presence of an excess of urea such as 7 M. However, significant interaction between oligo(dT) and PVAd was observed even at 7 M urea. This should not be surprising, since analytical affinity has predicted that a strong competitor to weaken the interaction between solute and an affinity ligand would be required to achieve zonal electrophoresis. Urea may not be the competitor, but since it can break the hydrogenbonding of the base-paired complex, urea will be effective for the weakening of the interaction between N and L, Le., a decrease in the association constant, K, in eq 3, in order to use affinity gel electrophoresis for analytical purposes. A decrease in the migration time of oligo(dT) was caused by a decrease in K , (eq 3). For preparative purposes, electrophoretic conditions, e.g., non-urea gels, should be selected in which the desired solute binds tightly enough to the affinity

A (CGE)

10

min

15

B (CAGE)

I _

10 0

2.0

4.0

6.0

8.0

Concentration of urea (M) Figure 7. Effect of the concentration of urea on the migration time of oiigodeoxynucieotldes. Conditionsare as in Figure 5 except for the concentration of urea and concentration of PVAd (0.01%).

.-cE E .-Im

2o

10

t

30

40

50

60

Capillary temperature ("C)

Figure 8. Effect of the capillary temperature on the migration time of oiigodeoxynucieotldes. Conditions are as in Figure 5 except for the capillary temperature.

ligand to be retained. We have used 7 M urea-gel throughout, because severe band-broadening was significantly reduced and high-performance base-specific separation was accomplished. We further examined the effect of capillary temperature on the base-specific separation, as shown in Figures 5B and 8. Migration time of both oligo(dA) and oligo(dT)decreased with capillary temperature. Oligo(dT)was more affected by temperature than oligo(dA). A decrease in viscosity of the surrounding gel-buffer with an increase in temperature28led to a decrease in migration time of oligo(dA). On the other hand, a decrease in migration time of oligo(dT1 was mainly caused by denaturation of complex between oligo(dT) and PVAd rather than the viscosity effect. Dissociation of hydrogen-bonding results in a decrease in the association constant, K,,in eq 3. Figure 5B demonstrates that all bands of oligo(dT) are electrophoresced with some bands of oligo(dA)at elevated temperatures. An electropherogramshowed (28) Guttman, A.; Cooke, N. J. Chrornatogr. 1991,559, 285-294.

60

L 15

"C (4-24 min)

rnin

Figure 0. Capillary affinity gel electrophoresisof (dA)rp-rsand (dV15. The gel contained 8% T and 5 % C (A) and 8% T, 5 % C, and 0.1% poiy(9-vlnyladenlne)with a molecular weight range of 10 000-30 000 (6). Capiilay temperature: 30 O C (A): 50 O C for 0-4 min and 60 O C for 4-20 min (B). Other conditions are as in Figure 4.

bands overlapping in comparison with Figure 4C, because a slight increase in the migration time of oligo(dT) caused by weak interaction remained. All the factors examined here strongly affected the electrophoretic behavior of oligo(dT) in the gel medium trapping affinity macroligand. Studies on quantification of these effects and optimization of separation conditions are now in progress. Of these parameters, capillary temperature is the most controllable to achieve high-performance specific base recognition. High-Performance Specific Base Recognition. Figure 5C demonstrates the effect of capillary temperatureprogrammingon electrophoreticbehavior in capillary affinity gel electrophoresis. A temperature program for 0-5 min at 30 O C and for 5-20 rnin at 50 O C was applied to the separation of a mixture of (dA)lz-ls and (dT)12-18. This program was actually performed as a gradient in which the temperature was gradually increased from 30 "C at 5 min and reached 50 "C after 8 min rather than as a stepwise change in temperature. The migration times of (dT)12-16, at which bands appeared, were significantly diminished compared with constant-temperature electrophoresis at 30 "C in Figure 5A. In addition, temperature-programming resulted in sharper bands compared with constant temperature. The situation is very similarto the band compression effect29 during gradient elution in HPLC, i.e., the band widths in gradient elution are diminished in comparison with those in isocratic elution. (dT)13-16 were separated completely from (dAh-18, but the ( d T h band partially overlappedthat of (dA)la. The (dTl14-16 bands were severelybroadened due to the powerful interaction with PVAd. A relatively simple mixture of oligodeoxynucleotides in the next section was separated by temperatureprogramming to show the performance of the base-specific separation by CAGE. Figure 9B demonstrates that the base-specific separation of a mixture of (dA)lz-18 and (dTI15 was achieved using capillary affinity gel electrophoresis with temperatureprogramming, whereas that without an affinity ligand was not, as shown in Figure 9A. The plate number of (dT)16was (0.5-3) X 106 plates/m in Figure 9B. The value was slightly reduced compared with the plate number calculated from Figures 4 and 9A, but much higher than that (by several tens of thousands) achieved by high-performance affinity chro(29) Snyder, L. R.; Saunders, D. L. J. Chrornatogr. Sci. 1969,7,195208.

ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 1992

matography.22 The resolving power and separation speed of capillary affmity gel electrophoresisin base-specific separation compared favorably with those of high-performance affinity chromatographyl3,22.24,26and conventional affinity gel electrophoresis.12,*4,16,17,23,25 For example, over 2 h is needed to complete the resolution of (A)lz-la using high-performance affinity chromatography.22 In conclusion,poly$-vinyladenine) was useful as an affinity macroligand for capillary affinity gel electrophoresis in achieving sensitive and high-resolution base-specific separation of oligodeoxynucleotides. Electrophoresis using urea gels with PVAd and temperature-programming was essential for analytical uses of CAGE. The high resolving power of CAGE will allow ita application to the selective recognition of DNA with specific base sequences in nucleic acid mixtures, using well-designed affinity macroligands including complementary base sequences.

1925

ACKNOWLEDGMENT We gratefully acknowledge support for this research by a travel grant from the Yoshida Foundation for Science and Technology. The present work was partially supported by a Grant-in-Aid for Scientific Research No. 04771913 from the Ministry of Education, Science, and Culture. RECEIVED for review December 23, 1991. Revised manuscript received April 3, 1992. Accepted May 26, 1992. Registry No. DBU, 6674-22-2;polydA, 25191-20-2;polyd?‘, 1925548-2;9-vinyladenine, 25086-81-1;9-(2’-chloroethyl)adenine, 20245-85-6; polyacrylamide, 9003-05-8; poly(9-vinyladenine), 26747-12-6;urea, 57-13-6.