Hybridization Properties of Support-Bound Oligonucleotides: The

Bioconjugate Chem. 2003, 14, 811−816

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Hybridization Properties of Support-Bound Oligonucleotides: The Effect of the Site of Immobilization on the Stability and Selectivity of Duplex Formation Kaisa Ketoma¨ki,*,† Harri Hakala,‡ Outi Kuronen,† and Harri Lo¨nnberg† Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, and PerkinElmer Life and Analytical Sciences, Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland. Received January 15, 2003; Revised Manuscript Received June 10, 2003

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 µm) polymer particles through C5-tethered thymine and N4-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.

INTRODUCTION

The so-called DNA-chip technology based on solidsupported high density oligonucleotide arrays is one of the most rapidly developing experimental techniques in molecular biology and biotechnology (1-3). An increasing number of applications dealing with analysis of gene expression (4-6), DNA polymorphism (7-10), RNA folding (11-13), and various enzyme-mediated detection methods (14-20) have gained interest. The key step in the construction of oligonucleotide arrays is immobilization of the oligonucleotide probes to a planar support. Short probes may be assembled from monomeric phosphoramidite building blocks in situ on the support (2126). On using longer probes, the arrays are usually obtained by postsynthetic covalent immobilization of prefabricated oligonucleotides to an appropriatedly derivatized support. Either an oligonucleotide bearing a functionalized sidearm (27-38) or a nucleophilic phosphorothioate linkage (39, 40) is reacted with an appropriate support-bound functional group, or an underivatized oligonucleotide is cross-linked to the support through its natural functionalities either photochemically (41) or by chemical reaction with a highly reactive activated surface (42, 43). Alternatively, noncovalent immobilization based on multiple electrostatic interactions of the polyanionic oligonucleotide with a polycationic support may be applied (44). Despite the continuously increasing number of applications of the array technique, the details of the molecular interactions occurring at the interface of the solution and solid phase are still rather poorly known (45-47). In particular, it is not clear how the site of attachment within an immobilized probe, and hence the orientation with respect to the support, affects the efficiency and selectivity of hybridization. Knowledge on such factors * To whom correspondence should be addressed. Phone +3582-3337639.Fax: +358-2-3336731.E-mail: [email protected]. † University of Turku. ‡ PerkinElmer Life and Analytical Sciences.

Scheme 1. Principle of the Sandwich Type Hybridization Assay

is, however, relevant, since covalent immobilization of prefabricated underivatized oligonucleotides evidently yields arrays where several different bonding modes are present, and even on using functionalized oligonucleotides, aimed at reacting through a single functional group, interference of other functional groups may lead to nonhomogeneous arrays. We have previously elucidated the factors affecting the efficiency of mixed-phase hybridization by using oligonucleotide-coated microscopic polymer particles (50 µm) as the solid phase and probes labeled with a fluorescent europium(III) chelate for quantification of the hybridization efficiency by a sandwich type assay (48-53). Accordingly, the PCR-amplified oligonucleotide forms first in solution a duplex with the fluorescently tagged probe and hybridizes then with the particle-bound probe (Scheme 1). The lanthanide ion emission is measured directly from a single particle. Special attention has been paid to the detection of point mutations (53), which plays an important role in population genetics (54) and in diagnostics of cancer (55) and viral diseases (26). It has been shown that oligonucleotide probes tethered to the support

10.1021/bc0340058 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/24/2003

812 Bioconjugate Chem., Vol. 14, No. 4, 2003

through their 3′-terminus quite well recognize a one-base mismatch present in the central or 5′-terminal region of a 10- to 12-mer support-bound probe. The hybridization efficiency is reduced by 1-2 orders of magnitude. By contrast, a mismatch at the penultimate site at the 3′end of the immobilized probe does not appreciably destabilize the duplex, and in some cases even a stabilization takes place. Studies on microarrays have also suggested that position of the mismatch near the terminus of the probe affects the duplex stability more than the identity of the mismatch (56). To learn how susceptible the hybridization actually is to conformational constrains that result from tethering to a solid support through various sites, four 12-mer oligodeoxyribonucleotides were coupled through four different sites to 50 µm polymer particles, and their hybridization with fully complementary 10- to 12-mer sequences, as well as with several 12-mer sequences containing a single-base mismatch at various sites, was quantified by a sandwich type assay (Scheme 1). A 35 atom long linker was used to tether the oligonucleotide from C5 of a thymidine residue to the support. It has been reported previously that on a polypropylene support the optimal spacer length is 40 atoms (57). EXPERIMENTAL PROCEDURES

Preparation of Oligonucleotide-Coated Microparticles. The particles used for the immobilization of the oligonucleotide probes were porous uniformly sized (50 µm) polymer particles made of a copolymer of glycidyl methacrylate (40%) and ethylene dimethylacrylate (60%) and functionalized with primary amino groups (1 mmol g-1) by reacting the particle-bound epoxy groups with diethylenetriamine (SINTEF). The surface area of the particles was 137 m2 g-1. These particles were coated with mercaptoalkyl tails by acylating the amino groups with 15-(4,4′-dimethoxytrityloxy)-12,13-dithiapentadecanoic acid in dry pyridine, using N-hydroxysuccinimide, N,N′-diisopropylcarbodiimide, and 4-(dimethylamino)pyridine as activators, and capping the rest of the amino groups with acetic anhydride in pyridine (58). The (4,4′dimethoxytrityl) loading obtained was 6-9 µmol g-1.The disulfide bond was then reductively cleaved with DTT (0.1 mol L-1 in MeOH, mildly basic conditions with Et3N), and the mercapto-group-coated particles were treated with 2-pyridyl disulfide-activated oligonucleotides overnight in water (200-400 µL). The UV-spectrophotometrically determined oligonucleotide loading was 1-2 µmol g-1. The unreacted mercapto functions were capped with maleimide in a 1:1 mixture of pyridine and ethanol (1 mL of 0.5 mol L-1 solution), washed, and dried (49). The 2-pyridyl disulfide-activated oligonucleotides were prepared as described below. Preparation of Oligonucleotides. The 2-pyridyl disulfide-activated oligonucleotides used in the preparation of oligonucleotide-coated particles were obtained as follows. Oligonucleotides bearing an aminoalkyl sidearm at desired sites were first assembled by the normal phosphoramidite strategy, using in appropriate places 5-[3-oxo-3-(N-trifluoroacetyl-6-aminohexyl)amino-1-propenyl]-2′-deoxyuridine or N4-(N-trifluoroacetyl-6-aminohexyl)-2′-deoxycytidine 3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as building blocks. The former building block was a product of Glenn Research, and the latter was prepared as described previously (59). The aminoalkyl-functionalized oligonucleotides were purified by ion-exchange HPLC on a SynChropak AX-300 column (4.6 mm × 250 mm, 6.5 µm; buffer A, 0.05 M KH2PO4 in 50% aqueous formamide, pH 5.6; buffer B, buffer A +

Ketoma¨ki et al.

0.6 M (NH4)2SO4: flow rate 1 mL min-1; a linear gradient from 0 to 50% buffer B in 40 min) and desalted. The functionalized oligonucleotides were converted to 2-pyridyl disulfide derivatives by acylating the side chain amino function with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP; product of PIERCE) in 70% aq MeCN in the presence of Et3N (100-200 µL). SPDP was used in 100-fold excess, and the stirring was continued overnight. The 2-pyridyl disulfide-activated oligonucleotides obtained were purified by RP-HPLC and desalted. The target oligodeoxyribonucleotides (O1-O27) were assembled by normal phosphoramidite strategy and purified by ion-exchange HPLC on a SynChropak AX300 column (4.6 mm × 250 mm, 6.5 µm; buffer A, 0.05 M KH2PO4 in 50% aqueous formamide, pH 5.6; buffer B, buffer A + 0.6 M (NH4)2SO4: flow rate 1 mL min-1; linear gradient from 0 to 60% buffer B in 30 min) and desalted. The preparation of the fluorescently tagged oligodeoxyribonucleotide probe 5′-d(CX5ATATCATCTTTGGTGT)3′ (F1), where X stands for N4-(6-aminohexyl) 2′-deoxycytidine bearing an amino-tethered photoluminescent lanthanide chelate, {2,2′,2′′,2′′′-[(4′-{4′′′-[(4,6-dichloro1,3,5-triazin-2-yl)amino]phenyl}-2,2′:6′,2′′-terpyridine6,6′′-diyl)bis(methylenenitrilo)]}tetrakis(acetato)eurupium(III), has been described earlier (49, 51). Hybridization Assays. Hybridization assays were carried out in a Tris buffer (50 mmol L-1, pH ) 7.5, containing 0.5 mol L-1 NaCl and 0.01% Tween 20). Typically 50 oligonucleotide coated particles (P1-P16 in Table) were shaken in 10 µL of buffer containing the target oligonucleotide (O1-O27) and the fluorescently tagged probe at a concentration of 17 nmol L-1. The mixtures were shaken in sealed tubes with rotamix (15 rpm) at 25 °C overnight. The particles were then rapidly washed with buffer, and 10 of the particles were subjected one after another to a time-resolved measurement of the fluorescence emission directly from the particle (48). The standard deviation ranged from 5 to 20%. As shown previously (49-53), the unspecific binding was always less than 1%. The emission signal was converted to the number of the fluorescently tagged probes attached to the particle with the aid of a calibration line obtained by determining the amount of europium(III) ions released from the particle by the DELFIA protocol (60), as described previously in more detail (51, 53). RESULTS AND DISCUSSION

Preparation of the Oligonucleotide-Coated Particles. The particles used as the solid support in the mixed-phase sandwich-type hybridization assays (Scheme 1) were uniformly sized (50 µm) porous particles of SINTEF that were made of a copolymer of glycidyl methacrylate (40%) and ethylene dimethylacrylate (60%) and functionalized with primary amino groups (1 mmol g-1) by reacting the particle-bound epoxy groups with diethylenetriamine. The resin-bound aminoalkyl sidearms were elongated by acylating the amino groups with 15-[(4,4′-dimethoxytrityl)oxy]-12,13-dithiapentadecanoic acid (58), and the terminal mercapto function was exposed by reductive cleavage of the disulfide bond (Scheme 2). 2-Pyridyl disulfide activated oligonucleotides, bearing the activated sidearm at various sites of the 12mer sequence, were then prepared as depicted in Scheme 3 and attached to the mercapto-derivatized particles via spontaneous disulfide bond formation in aqueous solution. Accordingly, the linker between the resin and the nucleic acid base is 36 and 32 atoms long on using immobilization through uracil and cytosine bases, re-

Hybridization of Solid-Supported Oligonucleotides Scheme 2. Particles

Preparation of Mercapto-Functionalized

Table 1. Oligodeoxyribonucleotide Sequences Attached to the Particlesa sequence on the particleb,c P1 P2 P3 P4 P5 P6 P7 P8

S-T*GA TTC ATC GCT-5′ S-TGA T*TC ATC GCT-5′ S-TGA TTC AT*C GCT-5′ S-TGA TTC ATC GCT*-5′ S-T*AG TCA GTC GTT-5′ S-TAG T*CA GTC GTT-5′ S-TAG TCA GT*C GTT-5′ S-TAG TCA GTC CTT*-5′

sequence on the particle P9 P10 P11 P12 P13 P14 P15 P16

S-T*GA CGA TCT CAT-5′ S-TGA C*GA TCT CAT-5′ S-TGA CGA TC*T CAT-5′ S-TGA CGA TCT CAT*-5′ S-T*GA TCT ACT GAT-5′ S-TGA T*CT ACT GAT-5′ S-TGA TCT AC*T GAT-5′ S-TGA TCT ACT GAT*-5′

a For the structure of the linkers employed, see Chart 1. b S stands for the polymer particle. c The nucleosidic unit used for tethering is indicated by an asterisk (N*).

spectively. Such linkers may be expected to allow rather unhindered hybridization, since the optimal length has been previously reported to be 40 atoms (57). Table 1 lists the oligonucleotide-coated particles (P1-P16) prepared. Relationship between the Site of the Immobilization Site and Duplex Stability. The binding efficiency of 27 oligodeoxyribonucleotides (O1-O27) to particles

Bioconjugate Chem., Vol. 14, No. 4, 2003 813

bearing a complementary sequence (P1-P16) was determined by a sandwich-type setup (Scheme 1). The 5′terminal region of these oligonucleotides was over 12 nucleotides fully or partly complementary to the supportbound probe, while a common 16 nucleotide long sequence at the 3′-terminus served to bind the fluorescently tagged probe, 5′-d(CX5ATATCATCTTTGGTGT)-3′ (F1), where X stands for a N4-(6-aminohexyl)-2′-deoxycytidine unit labeled at the amino group with a photoluminescent lanthanide chelate. The complementarity over 16 nucleotides is sufficient to warrant virtually quantative duplex formation between F1 and the target oligonucleotide (O1-O27) (51). The intensity of the emission signal measured directly from a single particle is over a wide range proprotional to the amount of the fluorescently tagged probed bound to the particle, since the quenching is negligible with photoluminescent lanthanide chelates (49-51). Table 2 records the affinity of 14 different target sequencies (O1-O14) to the sequence 3′-TGA TTC ATC GCT-5′ immobilized to the polymer particles in four different manners (P1-P4). The affinity is given as the amount of the target oligonucleotide hybridized to the support-bound probes divided by the total amount of the target oligonucleotide in the assay [n(bounds)/n(total)]. Among the target oligonucleotides studied, O1 and O2 are fully complementary with the particle-bound probe (P1-P4) over 12 and 11 adjacent bases, respectively, while O3-O5 all are complementary to P1-P4 over a sequence of 10 bases. All these oligonucleotides behave similarly: the affinity is markedly reduced when the probe is tethered to the particle through an internal (P2, P3) or 5′-terminal (P4) thymine base instead of the 3′terminal base (P1). The markedly lower hybridization

Scheme 3. 2-Pyridyl Disulfide Activation of Amino-Tethered Oligonucleotides

814 Bioconjugate Chem., Vol. 14, No. 4, 2003

Ketoma¨ki et al.

Chart 1. Linker Structures of the Oligonucleotide-Coated Particles

Table 2. Effect of the Site of Immobilization on the Hybridization Efficiency of Support-Bound Probes, P1-P4, at pH 7.5 and 25 °Ca O1 O2 O3 O4 O5 O6 O7 O8 O9 O10 O11 O12 O13 O14

Table 4. Effect of the Site of Immobilization on the Hybridization Efficiency of Support-Bound Probes, P9-P12, at pH 7.5 and 25 °Ca

target oligonucleotideb

P1

P2

P3

P4

5′-ACTAAGTAGCGA -[oligo]-3′ 5′-ACTAAGTAGCGT -[oligo]-3′ 5′-ACTAAGTAGCTT -[oligo]-3′ 5′-CTAAGTAGCGT -[oligo]-3′ 5′-TAAGTAGCGA -[oligo]-3′ 5′-AGTAAGTAGCGA -[oligo]-3′ 5′-ACTAATTAGCGA -[oligo]-3′ 5′-ACTAAGTATCGA -[oligo]-3′ 5′-AGTAAGTAGCGT -[oligo]-3′ 5′-ACTAATTAGCGT -[oligo]-3′ 5′-ACTAAGTATCGT -[oligo]-3′ 5′-AGTAAGTAGCTT -[oligo]-3′ 5′-ACTAATTAGCTT -[oligo]-3′ 5′-ACTAAGTATCTT -[oligo]-3′

0.28 0.27 0.19 0.24 0.23 0.33 0.07 0.06 0.17 0.07 0.06 0.07 0.03 0.03

0.08 0.08 0.07 0.07 0.03 0.06 0.03 0.03 0.07 0.03 0.02 0.02 0.00 0.00

0.05 0.03 0.02 0.03 0.02 0.02 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00

0.15 0.08 0.03 0.12 0.14 0.09 0.03 0.03 0.04 0.02 0.01 0.01 0.00 0.00

a

The hybridization efficiency is given as n(bound)/n(total) for the target oligonucleotide (O1-O8). b The abbreviation [oligo] stands for the sequence 5′-ACACCAAAGATGATATT-3′, in which the nucleotides given in italics were used to bind the fluorescently tagged oligonucleotide F1. The sequence engaged in duplex formation is indicated in bold. The mismatched base is underlined. Table 3. Effect of the Site of Immobilization on the Hybridization Efficiency of Support-Bound Probes, P5-P8, at pH 7.5 and 25 °Ca O15 O16 O17


P5

P6

P7

P8

5′-ATCAGTCAGCAA-[oligo]-3′ 5′-ATCAGTCAGCAT-[oligo]-3′ 5′-ATCAGTCAGCTT-[oligo]-3′

0.28 0.24 0.31

0.34 0.22 0.19

0.12 0.14 0.10

0.26 0.33 0.25

a The hybridization efficiency is given as n(bound)/n(total) for the target oligonucleotide (O9-O22). b See footnote b in Table 2.

efficiency of the 5′-immobilized probes compared to their 3′-bound counterparts could possible be attributed to steric hindrances resulting from interaction of the dangling end of O1-O5 and, hence, also the fluorescently tagged probe F1, containing five bulky chelates, with the support. The fact that tethering through an internal base moiety (P2, P3) retards the hybridization even more markedly than immobilization through the 5′-terminal base does not, however, lend support to this interpretation, but suggests that intrachain and to a lesser extent 5′-immobilization restrict the conformational flexibility of the probe more severely than the 3′-tethering, and hence the hybridization is retarded. Tables 3-5 summarize the corresponding data on the hybridization fully complementary oligonucleotides of three other probes, all immobilized in four different manners: hybridization of O15-O17 to P5-P8 (Table 3), O18-O22 to P9-P12 (Table 4), and O23-O27 to P13-P16 (Table 5). The results are very similar to those discussed above.The data in Tables 4 and 5 is consistent with the conclusions drawn above: the hybridization

O18 O19 O20 O21 O22


P9

P10

P11

P12

5′-ACTGCTAGAGTA-[oligo]-3′ 5′-ACTGCTAGAGTT-[oligo]-3′ 5′-ACTGCTAGAGAT-[oligo]-3′ 5′-TGCTAGAGTA-[oligo]-3′ 5′-CTGCTAGAGTT-[oligo]-3′

0.28 0.36 0.43 0.33 0.41

0.05 0.05 0.04 0.07 0.06

0.02 0.03 0.02 0.02 0.02

0.06 0.09 0.13 0.07 0.10


Table 5. Effect of the Site of Immobilization on the Hybridization Efficiency of Support-Bound Probes, P13-P16, at pH 7.5 and 25 °Ca O23 O24 O25 O26 O27


P13

P14

P15

P16

5′-ACTAGATGACTA -[oligo]-3′ 5′-ACTAGATGACTT -[oligo]-3′ 5′-ACTAGATGACAT -[oligo]-3′ 5′-TAGATGACTA -[oligo]-3′ 5′-CTAGATGACTT -[oligo]-3′

0.18 0.23 0.31 0.22 0.27

0.11 0.09 0.05 0.06 0.07

0.01 0.02 0.01 0.00 0.01

0.05 0.08 0.08 0.03 0.07


efficiency is decreased in the order P1 > P4 > P2, P3. Nevertheless, expections from this general trend appear to occur. Targets O15-O17 do not exhibit a higher affinity to the 3′-tethered probes (P5) than to the intrachain (P6, P7) or 5′-immobilized (P8) oligonucleotides. Another interesting question is how does the site of immobilization influence on the detection of a one-base mismatch at different parts of the probe. We have shown previously (53) that a one-base mismatch close to the 3′end of a 3′-immobilized probe does not noticeably destabilize the duplex, while a similar mismatch in the central or 5′-terminal part of the probe reduces the hybridization efficiency by 1-2 orders of magnitude. The results of the present study are consistent fairly well with those observations. For example, a point mismatch at the penultimate position of the 5′-end of target O6, i.e., a site that becomes close to the 3′-end of the support-bound probe upon hybridization, does not reduce the hybridization to a 3′-immobilized probe (P1), but increasingly does so on shifting the site of immobilization toward the 5′terminus (P2 f P3 f P4). When the mismatch is situated in the central part of the particle-bound probe, as on using O7 or O8 as a target, it becomes recognized by all the probes (P1-P4). Accordingly, while an intrachain tethering markedly reduces the efficiency of hybridization, it simultaneously allows rather reliable detection of a single base polymorphism at any site of the target. The affinities of the targets having a shorter complementary region (O9-O14) are too low to allow firm conclusions, but the behavior seems to be similar to that discussed above.

Hybridization of Solid-Supported Oligonucleotides

In summary, the results of the present study indicate that the selectivity of hybridization that may be achieved with a probe of a given length are markedly dependent on the site of immobilization within the probe. The 3′immobilization generally allows more efficient hybridization than 5′- or intrachain-tethering. Reliable detection of a one-base mismatch close to the terminus used for the immobilization is, however, difficult. In fact, intrachain tethering appears to allow a rather reliable detection of a point mutation at any site within the target. Random immobilization through various sites will probable make detection of point mutation impossible. ACKNOWLEDGMENT

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