簡便な遺伝子解析を志向した電気化学的分子ビーコン熊本大学大学院自然科学研究科准教授井原敏博 1 核酸分析法シグナルの種類 RI UV 発光電気化学システム形態固相単体微粒子均一溶液それぞれに特長、欠点がある。用途に合わせて選択される必要がある。 2 電気化学法の特長高感度選択的応答安価 3 電気化学法による従来の核酸分析 Published on Web 08/17/2004 DNA-PEG-DNA Triblock Macromolecules for Reagentless DNA Detection Chad E. Immoos,† Stephen J. Lee,‡ and Mark W. Grinstaff*,§ Departments of Biomedical Engineering and Chemistry, Metcalf Center for Science and Engineering, Boston UniVersity, Boston, Massachusetts 02215, Army Research Office, Research Triangle Park, O North Carolina 27709, and Department of Chemistry and Biochemistry, O O CaliforniaNPolytechnic State UniVersity, San Luis Obispo, California 93407 C ACTCACTGTGTCCCCGGTC P O O H Fe Received June 8, 2004; E-mail: [email protected] Electrochemical methods to detect specific nucleic acid sequences of hereditary diseases, genetic abnormalities, and viral or bacterial pathogens are of widespread importance to providing correct medical diagnosis and treatment. The advantages of electrochemicalbased hybridization assays for such DNA screening include rapid detection, sensitive electrochemical transducers, minimal power requirements, compatibility with microfabrication techniques, elimination of sample amplification, and low production costs.1-3 Several electrochemical detection schemes have been reported in the last 10 years. For example, electrocatalytic signal amplification approaches have been described by Barton, Thorp, and Bazan.4-7 Electrochemiluminescence assays have also been reported for the detection of specific DNA sequences.8 Recently, the electrochemical detection of DNA using immobilized molecular hairpins9-11 and single-stranded DNA12 have been reported. The sandwich assay is the most common design for electrochemical DNA sensors.13,14 This assay consists of three individual DNA components: an imIhara et al. NAR (1996) mobilized capture strand, a target strand, and a probe strand Ihara et al.reporter ChemComm containing a redox-active group. All(1997) three components must come together to elicit an electrochemical response at the electrode surface. Herein, we describe a simplified “two-piece” reagentless electrochemical assay for DNA detection that exploits a conformational change that occurs when a surface-immobilized, ferrocenelabeled oligodeoxynucleotide-poly(ethylene glycol) triblock mac- Scheme 1. Electrochemical Detection of Target Nucleic Acid Sequences Using a DNA Wrap Assay as Opposed to a Conventional Sandwich Assay Fc-DNAprobe-PEG-DNAcapture-SH-3! macromolecule used in this study was 5!-Fc-C6-GTACCACACCAA-(PEG)-GCACATAGAAGGCGA-C6-SH-3!. The melting temperature of the capture:target Grinstaff duplex et al. JACS (2004) duplex was 50.2 °C, the capture:probe was 48.1 °C, and the capture:target:probe duplex was 49.0 °C (1 µM DNA in 5 mM sodium phosphate, 50 mM NaCl, pH 7.0 buffer). Circular dichroism spectra of the target DNA strand in the presence of capture strand, probe strand, or both show transitions at wavelengths characteristic for B-form DNA When the 5!-Fc-DNA-PEG-DNA-SH-3!macromolecule is im- 4 この研究のねらい均一溶液中でターゲット選択的な電気化学応答を可能にしたいターゲットへの結合電流シグナルの変化モレキュラービーコン（MB）のように近づいたときに消光させる工夫が必要（電気化学的応答を抑え込むしかけ） 5 βCyD - DNA conjugate HO 5' 3' O O TGTGTCCCCGGTCTG P O O N H O C O OH O O OH OH HO OH O O OH HO O OH O OH O S OH S HO O OH OH OHOH OH OH O O O O HO O OH 6 Recognition & Detection redox potential ? current intensity ? Fc-DNA βCyD-DNA target Mut Fc-DNA βCyD-DNA target WT OH O R Fe Ferrocene + HO O O OH HOOH O OH O HO O O OH OH OH HO OH O HO OOH OH OH OH OH O O O OOH HO O OH βCyD inclusion complex K = 102 - 103 M-1 ΔG° ≈ 4 kcal mol-1 7 βCyD - DNA conjugate synthesis, 1 N SS O O CO N O H2N O O P O O SPDP NNN...NN O O S OO OH O HO O O OH HOOH O OH O HO O O OH OH OH HO OH O HO OOH OH OH OH OH O O O OOH HO O OH TsCl Pyridine HO O O OH HOOH O OH O HO O O OH OH OH HO OH O HO OOH OH OH OH OH O O O OOH HO O OH N S S O C N H O O P O O NNN...NN CH3 SH O HO O O OH HOOH O OH O HO O O OH OH OH HO OH O HO OOH OH OH OH OH O O O OOH HO O OH 1. CS(NH2)2 2. NaOHaq HO O O O OH O HO OH OH OH HO O OH O OH O S O OH HO S OH O OH HO OH HO HO OH O O O O OH O HO O C N H O O P O O NNN...NN βCyD(8)ODN 8 Fc - DNA conjugate synthesis O N OH O N-Hydroxysuccinimide HOOC Fe O O N O C Fe O ACTCACTGTGTCCCCGGTC O O P O O NH2 ACTCACTGTGTCCCCGGTC O O P O N C H O O Fe FcODN 9 UV melting Fc-DNA βCyD-DNA 5’ ACTACACTGTGTCCCCGGTCTGGAAAC TGATGTGACACAGGGGCCAGACCTTTG TPMT gene βCyD(8)ODN Normalized OD tandem duplex Tmax / °C 37.4, 73.5 18.7, 73.9 280 300 320 340 360 19.7, 73.4 Temperature / K 24.1, 73.5 1 μM DNA, 10 mM Phosphate buffer (pH 7), 1 M NaCl 10 Thermodynamic parameters Normalized absorbance at 260 nm Normalized absorbance at 260 nm βCyD(8)ODN R = 0.99996 270 280 290 300 310 R = 0.99996 290 Temperature / K 300 310 320 330 Temperature / K neighbor ODN ΔΗ° / kcal mol-1 ΔS° / cal mol-1 K-1 ΔG° / kcal mol-1 K298 / M-1 unmodified -42.1 -117 -7.36 2.50 x 105 Fc-ODN -60.4 -167 -10.6 6.52 x 107 1 μM DNA, 10 mM Phosphate buffer (pH 7), 1 M NaCl 11 Electrochemistry 5’ ACTACACTGTGTCCCCGGTCTGGAAAC TGATGTGACACAGGGGCCAGACCTTTG CV with comb-shaped microelectrode SWV with disc electrode 10 nA 100 nA -0.2 0 0.2 0.4 0.6 Potential / V vs. Ag/AgCl 100 μM DNA, 10 mM Phosphate buffer (pH 7), 0.5 M KCl 0 0.1 0.2 0.3 0.4 0.5 0.6 Potential / V vs. Ag/AgCl 20 μM DNA, 10 mM Phosphate buffer (pH 7), 0.5 M KCl 12 Normalized absorbance at 260 nm SNP discrimination 5’ TGGAAAC ACNTTTGGTAAAGTAGAGTTGG TPMT genes G-N G-A G-T G-G G-C CATTTCATCTCAACC A T G 10 nA CV measurement C 280 290 300 310 320 330 340 350 Temparature / K 1 μM DNA, 10 mM Phosphate buffer (pH 7), 0.5 M KCl 0.1 0.2 0.3 0.4 0.5 0.6 Potential / V vs. Ag/AgCl 20 μM DNA, 10 mM Phosphate buffer (pH 7), 0.5 M KCl 13 Conclusions • DNA末端に修飾したFcとβCyDは target上で包摂錯体を形成し、二本鎖を大きく安定化した。 • βCyDによる包摂によりFcの電気化学シグナルは著しく抑制された。 • targetの塩基配列に依存した電気化学シグナルを得ることができた。 local minimum structure optimized by AMBER* 14 MB & ECMB 15 想定される業界創薬基礎研究機器開発医療・診断機器開発 16 本研究に関する知的財産権発明の名称 DNAコンジュゲートを利用した核酸の電気化学的検出法出願番号出願手続き中出願人熊本大学発明者井原敏博 17 本研究に関する問い合わせ先熊本大学瀬戸英昭コーディネーター（文部科学省、産官学連携コーディネーター） Tel: 096-342-3247 Fax: 096-342-3239 E-mail: [email protected] 18