Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-08
2002-04-09
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S050000, C422S068100, C536S025300, C536S025320, C536S026600
Reexamination Certificate
active
06368807
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a compound which is favorably employable as an electrochemically active threading intercalator in a procedure of analyzing oligonucleotides or polynucleotides such as DNA fragments.
BACKGROUND OF THE INVENTION
In the gene analysis in the fields of biochemistry and clinical test, the detection of a DNA or its fragment having a specific base sequence is performed by way of a hybridization method, particularly Southern hybridization method (Southern blotting method). Southern hybridization is performed using a radioisotope (RI) label. The conventional analytical methods using radioisotope label such as Southern hybridization method are disadvantageous in that they need troublesome radioisotopes.
A Southern hybridization method using a fluorescent label in place of a radioisotope label is also known. This method is superior to the method using RI in safety and rapidness. Therefore, DNA chips comprising a substrate such as a slide glass or a silicone plate and a great number of oligonucleotide or polynucleotide molecules fixed onto the substrate are now commercially available for the use in the fluorescence detection systems. However, the fluorescence detection system has other disadvantageous features, that is, the fluorescent label is. gradually faded out under irradiation of stimulating rays; a specifically designed fluorescence-measuring apparatus should be installed; and an amount of a fluorescent label is restricted because internal quenching takes place
Recently, a new system for detection of DNA fragments which utilizes an electrode sensor onto which a group of probes comprising oligonucleotide molecules or polynucleotide molecules are fixed has been proposed in Japanese Patent Provisional Publication No. H9-288080 and Preprint of 57th Conference of Analytical Chemistry, pp. 137-138 (1996). In this system, an electrode which has an output terminal and further has he probe molecules fixed onto its surface is brought into contact with a DNA sample in an aqueous medium in the presence of a threading intercalator, and an electric current produced by applying an electric voltage between the electrode and another electrode introduced in the aqueous medium is measured.
As the threading intercalator, an electroconductive ferrocene-containing compound having oxidation-reduction activity (redox activity) which has the following chemical structure and can be specifically bonded to a hybrid or hybridized DNA is known:
The above-mentioned electrochemical detection system utilizing an electrode sensor is advantageous in easily detecting the hybrid DM structure on real-time basis. No fading-out takes place.
The above-illustrated conventional electroconductive threading intercalator has a structure comprising a core portion of a naphthalene-diimide cyclic group, a pair of linker porions each of which is attached to each of the two ends of the core portion, and a pair of electroconductive ferrocene moieties each of which is attached to other end of each linker. The ferrocene moiety has an oxidation-reduction activity and a conjugated system in which electrons freely move.
In the procedures for detecting DNA fragments complementary to the probe molecules fixed on the electrode, the amount of electric current produced by the application of electric potential to the electrode essentially depends on the nature of an electroconductive threading intercalator, though in part depends on the natures of probe molecules and DNA fragment samples, and the ionic concentration of the buffer solution employed in the detection procedure. In the use of the conventional threading intercalator of the above-mentioned formula, a peak electric current is observed when an electric potential in the range of approx. 450 to 620 mV is applied. Therefore, in the detection procedures utilizing the conventional threading intercalator, an electric potential of approx. 450 mV or higher should he applied.
The electric potential of approx. 450 mV or higher is relative high for current detection devices. Accordingly, the cost for producing the detection devices for the electrochemical analysis of DNA fragments is relatively high. Moreover, if the probe molecules are attached to the electrode surface by weak bonding such as electrostatic bonding, the probe molecules are apt to be released from the electrode when a high electric potential is applied to the electrode. The release of the probe molecules from the electrode adversely effect to the detection sensitivity and detection accuracy. Particularly, the easy release of the probe molecules from the electrode adversely effect when the DNA chip is repeatedly employed in the detection procedures after the temporarily fixed DNA fragment samples and threading intercalator are removed.
Accordingly, it is an object of the invention to provide an electroconductive threading intercalator which is favorably employable in the electrochemical method for detecting polynucleotide samples or oligonucleotide samples (such as DNA fragments) by means of a DNA chip comprising an electrode and probe molecules (such as nucleo-tide derivatives or their analogues)
Specifically, it is an object of the invention to provide an electroconductive threading intercalator which is capable of working in the electrochemical detection procedure at a low electric potential applied to the electrode
SUMMARY OF THE INVENTION
The present invention resides in a compound having the formula (1):
Ea—La—X—Lb—Eb (1)
in which each of Ea and Eb independently is a group having oxidation-reduction activity and having a conjugated system in its group; X is a divalent cyclic group; and each of La and Lb independently is a group which does not form a conjugated system in combination with the conjugated system of each of Ea and Eb and at least one of which has a site imparting water solubility to the compound or a site that is convertible into a site imparting water solubility to the compound.
In the above-mentioned formula, it is preferred that Ea is the same as Eb, and La is the same as Lb. The main chain of La—X—Lb preferably contains 10 to 100 atom, more preferably 15 to 70, most preferably 20 to 50, which are counted along the shortest connection route from Ea to Eb. For the sake of reference, the main chain of the aforementioned conventional threading intercalator has 32 carbon atoms.
The compound of the formula (1) preferably has the following formula (2):
Ea—L1a—L2a—X—L2b—L1b—Eb (2)
in which each of Ea and Eb independently is a group having oxidation-reduction activity and having a conjugated system in its group; x is a divalent cyclic group; each of L1a and L1b independently is a group which does not form a conjugated system in combination with the conjugated system of each of Ea and Eb; and each of L2a and L2b independently contains a linking group having a site imparting water solubility to the compound or a site that is convertible into a site imparting water solubility to the compound.
It is preferred that each of Ea and Eb of the formulas (1) and (2) independently a group having oxidation-reduction activity which is selected from the group consisting of a metallocene moiety, a 2,2′-bipyridine complex moiety, a cyclobutadiene moiety, a cyclopentadiene moiety, a 1,10-phenanthroline moiety, a triphenylphosphine moiety, a cathecol amine moiety, and a biologen moiety. Any of these moieties may have one or more substituents.
In the formula (2), it is preferred that each of L1a and L1b independently is a hydrocarbyl group which may have one or more substituents. The hydrocarbyl group preferably has 1 to 6 carbon atoms in its main chain, More specifically, it is preferred that each of L1a and L1b independently is an alkylene group having 1 to 6 carbon atoms or an alkenylene group having 2 to 6 carbon atoms. Each group may have one or more substituents.
In the formula (2), it is preferred that each of L2a and L2b independently is a linking group containing an atomic element other than carbon element. It is particularly preferred that each
Makino Yoshihiko
Takagi Makoto
Takahashi Kazunobu
Takenaka Shigeori
Yamashita Kenichi
Fuji Photo Film Co. , Ltd.
Reed Smith LLP
Riley Jezia
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