ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 5, pp. 531–533. © Pleiades Publishing, Inc., 2007. Original Russian Text © A.N. Sinyakov, E.V. Kostina, G.A. Maksakova, O.A. Baturina, V.A. Ryabinin, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 5, pp. 571–573.
LETTERS TO THE EDITOR
Oligonucleotide Conjugates with Minor Groove Ligands as Probes for Hybridization Microarray Chips A. N. Sinyakov, E. V. Kostina, G. A. Maksakova, O. A. Baturina, and V. A. Ryabinin1 Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrentieva 8, Novosibirsk, 630090 Russia Received April 12, 2007; in final form, April 17, 2007
Abstract—-A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3')-probes: GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with the Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of AC and GT mutations in the binding site (imperfect duplexes) or a CG pair (perfect duplex) affect the change in fluorescence level to a considerably lesser degree. Key words: conjugates, microchip, minor groove binder, oligonucleotides, point mutation, single nucleotide polymorphism DOI: 10.1134/S1068162007050111
Oligonuclrotide hybridization chips are a convenient tool for determination of point mutations [1, 2].2 A prerequisite for a successful analysis is the site-specificity of the probe binding to the target DNA, which is determined to a substantial degree by the differences in stabilities of perfect and imperfect duplexes. One of problems is that the mutations close to the end of an oligonucleotide duplex do not affect substantially the stability of the duplex and do not allow significantly discriminate the perfect and imperfect duplexes [3, 4]. To overcome this obstacle, we proposed to use as probes the oligonucleotides bearing covalently linked site-specific minor groove ligands at the 5'- or 3'-ends. The most apt candidates for this role are the sequence-specific hairpin oligocarboxamides derived from N-methylpyrrol, N-methylimidazole, and β-alanine [5]. In this work, we studied a basic potential of this approach for the microchip design. As a model, we synthesized oligonucleotides bearing an aminohexyl residue at the 5'-end and a phosphate group at the 3'-end with an attached hairpin minor groove ligand [6] composed of two tripyrrolcarboxamide residues joined by an aminocaproyl residue (MGB). 1
Corresponding author; phone: +7 (3832) 330-4653, e-mail:
[email protected]. 2 Abbreviations: Cy3, a fluorescent cyanine dye; MGB, a minor groove ligand composed of two tripyrrolcarboxamide residues joined by an aminocaproyl residue.
Preliminary experiments showed that the use of 14−16-bp oligonucleotide probes is ineffective, probably, due to insufficient differences in stabilities of modified and unmodified probes. Therefore, we used shorter probes (1)–(4) of seven bases long. The mutation was shifted one base downstream rather than was located at the end of the oligonucleotide duplex. The spot location on the microchip is shown in the figure. Probes (1)–(4) as well as oligonucleotide (5) used as a printing control are located successively in four copies. The microchips were printed on glass aldehydebased slides according to the procedure [7]. Oligonucleotides (6) and (7) of 12-bp long containing a fluorescent cyanine dye at their 5'-ends were chosen as targets. Additional bases were introduced into the molecules in order to reduce the effect of the fluorophore on the formation of an oligonucleotide duplex. Thus, the hybridization of oligonucleotide probes and labeled oligonucleotides on the chip had to give duplexes containing A•T-, A•C-, G•T-, and G•C-pairs either unmodified or modified with the minor groove ligand (see the figure). The hybridization of fluorescently labeled oligonucleotides and probes was carried out at 25°ë for 40 min followed by successive washing of the slide with buffer solutions described in [7] and cooled down to 0°ë. The slide was scanned on an Express 2.0 scanner (Perkin Elmer) using an exciting laser with a wavelength of 543 nm. Analysis of the image and plotting of the his-
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5' 3' NH2(CH2)6-GACAAGAp
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NH2(CH2)6-GACAAAAp
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NH2(CH2)6-GACAAGAp-MGB
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NH2(CH2)6-GACAAAAp-MGB
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Cy3-AAAAAAA-(CH2)6NH2
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Cy3-ACTAATTTTGTC
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MGB =
H N
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tograms were achieved using the Scanarray Express program, which was a part of the scanner software. Hybridization patterns and the corresponding histograms of chip probe binding with target oligonucleotides (6) and (7) are shown in Figs. 1‡ and 1b, respectively. One can see that the A•T-specific minor groove ligand increases the stability of the duplex containing an A•T pair nearly fourfold, which is reflected in the hybridization pattern (Fig. 1‡). The fluorescence level of duplexes containing a G•T pair is lower in compari-
H N
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son with the perfect duplex bearing an A•T pair, and the introduction of a ligand does not cause a substantial change in the spot fluorescence level. In the case of Cy3-ACTAATCTTGTC(7) used as a target, the hybridization pattern changes: the most intensive fluorescence is observed for the spots corresponding to the unmodified duplex containing a G•C pair. The presence of a hairpin ligand in the probe only slightly affects the fluorescence level, which is explained by the ligand inability to be incorporated due
(a)
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(A · T) ~ MGB (G · T) ~ MGB
(A · ë)
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(A · ë) ~ MGB (G · ë) ~ MGB
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Fig. 1. A microchip image after hybridization of target oligonucleotides (a) Cy3-ACTAATTTTGTC (6) and (b) Cy3-ACTAATCTTGTC (7) with probes (1)–(4). The histograms show relative intensities of spot fluorescence of duplexes containing and lacking a hairpin ligand, which containA•T-, G•T-, A•C-, or G•C pairs in the mutation point (a mean value of four spots). (c), the probe positions on the microchip. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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to steric hindrances created by the guanine amino group located at the bottom of the minor groove [5]. The duplex containing an A•C pair is less stable, and the spot fluorescence is about 2.5 times lower than that of the G•C-containing duplex. The introduction of a ligand into the A•C containing duplex results in a small increase in the fluorescence level, which may be explained by a partial stabilization of the imperfect duplex due to ligand incorporation into the minor groove. However, the minor groove ligand, which can be site-specifically incorporated, affects the duplex stability and, hence, the spot fluorescence most intensively. In the example given above, the most significant effect was revealed for the perfect A•T duplex stabilized by an A•T specific minor groove ligand, whereas duplexes containing A•C and G•T mutations in the binding site (imperfect duplexes) or a G•C pair (perfect duplexes) are substantially less affective. One can assume that the use of oligonucleotides bearing minor groove ligands that manifest a high site specificity (composed of N-methylimidazole, β-alanine, and N-methylpyrrol residues) as probes would enhance their discriminating capacity, which would be useful for the design of hybridization microchips and can be applied for the structures other than probes with terminal mutations.
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ACKNOWLEDGMENTS The work was supported by CRDF, project no. 2652-NO-05, and Russian Foundation for Basic Research, project no. 05-08-01465-a. REFERENCES 1. Kwok, P.Y., Annu. Rev. Genomics Hum. Genet., 2000, vol. 1, pp. 235–258. 2. Khomyakova, E.B., Livshits, M.A., Sharonov, A.Yu., Prokopenko, D.V., and Mirzabekov, A.D., Mol. Biol., 2003, vol. 37, pp. 726–741. 3. Urakawa, H., Noble, P.A., El Fantroussi, S., Kelly, J.J., and Stahl, D.A., Appl. Environ. Microbiol., 2002, vol. 68, pp. 235–244. 4. Urakawa, H., El Fantroussi, S., Smidt, H., Smoot, J.C., Tribou, E.H., Kelly, J.J., Noble, P.A., and Stahl, D.A., Appl. Environ. Microbiol., 2003, vol. 69, pp. 2848–2856. 5. Dervan, P.B., Benjamin, S., and Edelson, B.S., Curr. Opin. Struct. Biol., 2003, vol. 13, pp. 284–299. 6. Ruabinin, V.A., Vutorin, A.S., Elen, K., Denisov, A.Yu., Rushnui, D.V., and Sinuakov, A.N., Bioorg. Khim., 2005, vol. 31, pp. 159–166; Rus. J. Bioorg. Chem., 2005, vol. 31, pp. 146–152. 7. Ryabinin, V.A., Shundrin, L.A., Kostina, E.V., Laassri, M., Chizhikov, V., Shchelkunov, S.N., Chumakov, K., and Sinyakov, A.N., J. Med. Vir., 2006, vol. 78, pp. 1325–1340.
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