.:) 1991 Oxford University Press

Nucleic Acids Research, Vol. 19, No. 15 4097-4102

Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy Akira Murakami, Misuzu Nakaura, Yuna Nakatsuji, Shunji Nagahara, Qui Tran-Cong and Keisuke Makino Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received May 29, 1991; Accepted July 10, 1991

ABSTRACT Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides.

INTRODUCTION Identification of gene sequences of microorganisms such as HIV and vibrio cholera can be achieved by means of sequence specific poly- and oligonucleotides as specific probes (1). This method, so called DNA-Probe method, attracts interests of physicians and molecular biologists as well as analytical chemists (2). Despite of its high potential to make diagnoses of diseases simple and reliable, DNA-Probe method has not been widely spread to researchers. There exist two major complicated problems which prevent DNA-Probe method from wide adoption to the diagnoses. They are (a) the usage of radioisotopes, and (b) the bound/free (B/F) separation procedures. Though radioisotopes such as 32p and 35S serve high sensitivity and reliability on monitoring genes, they are hazardous to researchers and environments. In order to avoid the usage of radioisotopes, enzyme-labeled DNA-Probes (3-6), for example, have been developed and as low as several attomols of DNA could be detected by the fluorometric method (6). To get informations from DNA-Probe after hybridization, it is necessary to efficiently separate unbound (free) DNA-Probe from

DNA-Probe bound to the target genes immobilized on the solid matrixes such as nitrocellulose membranes and hydrophilic beads. This series of procedures, called B/F separation, consists of multisteps and is time-consuming. In this paper, the methodology to monitor the hybrid formation in solution without B/F separation using fluorescent-labeled oligonucleotides is described. If some sort of labeled DNA-Probe gives rise to the characteristic change in its spectrum by being incorporated into a hybrid, the probe can be an excellent tool to detect the duplex formation, namely the presence of the target DNA sequence in solution. Electron spin resonance spectroscopy (ESR) is one of the candidates. ESR reflects changes of molecular motion of a spin-label as a change both in spectrum and in rotational correlation time. By now, several types of spin-labeled oligonucleotides have been developed and their characteristic behaviors in hybrid solution have been studied (7-10). In this methodology, detectable limits of amounts of hybrid in solution is as low as 10-'lmol. In this study, the fluorescence polarization spectroscopy, which is another candidate, was employed. This methodology was originally applied for detection and analysis of oligomeric protein dissociation and is highly sensitive and convenient (11,12). Here, fluorescein-labeled oligonucleotides, F-Probe, were utilized as the labeled-DNAProbe, and the fluorescence anisotropy, r, was measured in solution containing oligonucleotides. The hybrid formation between F-Probe and a single stranded DNA can be monitored by the change in r values, because F-Probes as well as the hybrids exhibit characteristic r values depending on their molecular weight, namely depending on their rotational correlation times.

EXPERIMENTAL Materials Fluorescein isothiocyanate (FITC, type 1) and fully protected nucleoside hydrogen phosphonates (triethylammonium salt) were purchased from Sigma Chemical Company. Fully protected nucleoside phosphoramidites and controlled pore glass beads (CPG) having 5'-di-p-methoxytrityl thymidine (DMTr-Thd) were purchased from Milligen Biosearch Inc. Other reagents were purchased from Wako Pure Chemicals, Inc. (Japan) and used without further purification.

4098 Nucleic Acids Research, Vol. 19, No. 15 (14,15) on controlled pore glass beads. The beads carrying ltmol of DMTr-Thd (72mg) were transfered to a gas-tight syringe (lmL) and dried in vacuo. The beads were treated with 2.5% dichloroacetic acid in methylene chloride for 30sec at room temperature followed by washing with acetonitrile. DMTrthymidine hydrogen phosphonate (0. lmL, 0.2M in pyridine/acetonitrile (1/1, v/v)) was added to the beads and the mixture was allowed to shake for lOsec followed by the addition of pivaloyl chloride (0. lmL, IM in pyridine/acetonitrile (1/1, v/v)). The mixture was agitated on a vortex mixer for 3min at room temperature. After the condensation step was repeated twice, the beads were washed sequentially with acetonitrile/pyridine (1/1, v/v) and acetonitrile, and dried. Diamine solutions (ethylenediamine (EDA) in CCl4, 1.5M, 0.4mL; hexamethylenediamine (HMDA) in CCl4; 0.5M, 0.4mL) were added to the beads and allowed to react for 20min. After the oxidation step was repeated twice, the beads were washed with CC14 and acetonitrile, and dried in vacuo. (16) FITC solution (0.5mL, lOmM in lOOmM-carbonate buffer (pH9)-DMF (20%, v/v) solution) was added to the beads and allowed to react in the dark at room temperature for 48h. Being washed sequentially with the carbonate buffer/DMF solution and acetonitrile, the beads were treated with concentrated ammonium hydroxide solution at 55°C for 8h. The supernatant combined with the washings of the beads was evaporated to dryness and the residue was subjected to HPLC purification.

Assay system Absorption and emission spectra were obtained in 0.1M phosphate buffer (pH7.0) with the final concentration of 2.5,uM. The solution containing both a fluoresein-labeled oligonucleotide (final concentration: 2.5ytM) and an oligonucleotide (final concentration: 2.51M) were once maintained at 85°C for 3min in a water bath and then gradually cooled down to 20°C. UVmelting curves of the solutions were obtained by a UV spectrometer (UV-260, Shimadzu Co., Japan) equipped with an automatic thermal controller. The increasing rate of the temperature was set to be 0.3°C/min. High performance liquid chromatography (HPLC) system (CCPM, Tosoh, Japan) equipped with a reversed phase column (Ultron N-C18, Shinwakako Inc., Japan) was used to purify labeledoligonucleotides with a linear gradient of acetonitrile (low: 5 % acetonitrile in lOOmM triethylammonium acetate (TEAA), high: 50% acetonitrile in 100)mM TEAA). Fluorescence spectra were obtained on a fluorescence spectrometer (RF-5000, Shimadzu Co., Japan) equipped with a set of polarizing plates. The fluorescence was measured at 520nm using an excitation wavelength of 480nm.

Preparation of fluorescein-labeled oligonucleotides Oligothymidylates t(n - 1)merj were synthesized by a phosphoramidite method (13) or by a hydrogen phosphonate one B

B

0-P-O4O0CPG

DMTr-

B

H-Phosphonate monomer

B

B

-P-- 0

DMTr- OJ

Acetonitile/pyrdine

-C-OPG O -2 O

H

CH2CH2CN

CH2CH2CN H2N

B

DMTr-O

B

B

04

B

F0PFrCO~OVV '*4 0 -2 CH2CH2CN NH(CH2),NH-CS-NH -®

~~~~DMF/

B

B

H 0JO-POJO-PQj

IOH

ON H(CH2)pNH-CS-NH

n-2 -.

Fig. 1. Synthetic scheme of fluorescein-labeled oligonucleotides.

B

B 0

DMTr-

O-P-O

O-P

Carbonate buffer CH2CH2CN

NH(CH2)pNH2

Ammonium hydroxide B

CC14

(CH2)p NH2

T2EF: n=2, p=2 T5EF: n=5, p=2 T11EF: n=11, p=2 T2HF:n=2, p=6 T5HF: n=5, p=6 T1 HF: n=l 1, p=6

F:fluorescein

OiPG

Nucleic Acids Research, Vol. 19, No. 15 4099

Preparation of fluorescein ethylamine (EA-F) and fluorescein hexylamine (HA-F) EA-F and HA-F were synthesized according to the reported procedure (17). For EA-F, 1ItL of 70% aqueous ethylamine solution was added to FITC solution (500SL, 30mM in DMF) and the resulting solution was allowed to react at room temperature in the dark for 24h. For HA-F, 0. lyL of hexylamine solution was added to FITC solution (lmL, lOmM in lOmM-carbonate buffer (pH 9)/20% DMF solution) and the solution was allowed to react at room temperature in the dark for 60h. The products were isolated by PAGE followed by HPLC purification. Fluorescence spectroscopic studies Fluorescence anisotropy (r) was calculated as r = [IVV-IVH(G)]/[Ivv + 2IVH(G)] ( G = IHV/IHH)

(1)

where Iw and IVH represent the emission intensities parallel and perpendicular to the polarization direction of excitation, respectively, and G value is a correction factor for the transmission efficiency of the emission monochromator. The samples (0. 1AM) were excited at 480nm and the fluorescence was monitored using a cutoff filter (