Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-26
2001-01-30
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C436S172000
Reexamination Certificate
active
06180350
ABSTRACT:
FIELD OF THE INVENTION
This application relates to compositions, methods and apparatus for the photo-electrochemical detection of nucleic acid of a defined sequence. In particular, one aspect of the invention features a device having an electrode, a first nucleic acid, a source for photons and a charge monitor. The first nucleic acid is covalently bound to the electrode with electron donor nucleotides proximal to the electrode and a modified nucleotide adjacent to the donor nucleotide. The modified nucleotide has a base with a functional group capable of receiving or donating electrons. The first nucleic acid donates or receives electrons from the electrode upon receiving photons. The rate at which the electrode receives or donates electrons with the first nucleic acid is different when the first nucleic acid is hybridized and when the first nucleic acid is unhybridized. The charge monitor measures the rate at which the electrode receives or donates electrons to determine the presence or absence of a second nucleic acid capable of forming a hybridization product with the first nucleic acid.
BACKGROUND OF THE INVENTION
The following definitions are provided to facilitate an understanding of the invention.
The term “target” or “target molecule”, in a diagnostic sense, refers to a molecule of interest, i.e. the molecule whose presence one wants to know. This application uses the term “signal nucleic acid” to refer to the target, if such target is present in sufficient quanities to produce a measurable response. However, the term “signal nucleic acid” also refers to nucleic acid that may be generated in the presence of target. By way of example, without limitation, signal nucleic acid may be generated in the presence of non-nucleic acid targets or nucleic acid targets through nucleic acid amplification reactions such as polymerase chain reactions (PCR) and RNA-dependent RNA polymerase reactions, such as Q-Beta replicase.
Nucleic acid antibody reactions are characterized by processes which are called SELEX (NexStar Pharmaceuticals, Inc., Boulder CO). These processes describe the manner in which nucleic acids may be selected for affinity to non-nucleic acid targets, such as proteins and other molecules of interest. The selected nucleic acids can be used as ligands for affinity reactions in the manner similar to antibodies.
Nucleic acid hybridization reactions are based on the affinity exhibited by complementary strands of nucleic acid. Nucleic acid target molecules can be identified by the binding of such target to a complementary ligand nucleic acid.
Assays based on nucleic acid antibodies and nucleic acid hybridization tend to be time consuming and complex. There exists a need for photo-electrochemical detection of signal nucleic acid.
SUMMARY OF THE INVENTION
This application relates to compositions, methods and apparatus for the photo-electrochemical detection of nucleic acid of a defined sequence. One embodiment of the present invention is a device for detecting the presence or absence of a signal nucleic acid. The device has comprising an electrode and a first nucleic acid covalently bound to the electrode. The first nucleic acid has two or more donor nucleotides capable of donating an electron. The donor nucleosides have a position in the first nucleic acid where one donor nucleotide is proximal to the electrode. The first nucleic acid has a modified nucleotide adjacent to one of the donor nucleosides. The modified nucleotide is capable of receiving an electron from said donor nucleotides upon photo-excitation and maintaining the electron for a first period of time when the first nucleic acid is unhybridized and a second period of time when the first nucleic acid is hybridized to the signal nucleic acid. The first and second periods are different. The electrode is in communication with the first nucleic acid to receive and donate electrons. The device further comprises a photon source for emitting photons onto the first nucleic acid. A charge monitor is in communication with the electrode for measuring the charge on the electrode or current flowing through the electrode as the first nucleic acid receives photons from the photon source which charge on said electrode or current is different in the presence of signal nucleic acid. The difference is indicative of the presence or absence of the signal nucleic acid.
Preferably, the modified nucleotide comprises a base and a functional group, and the donor nucleotides are more easily oxidized than the base of the modified nucleotide. Of the bases which comprise the first nucleic acid, guanine is more easily oxidized than adenine. Adenine is more easily oxidized than uracil, thymine, and cytosine. A preferred two or more donor nucleotides comprise three or more guanosine nucleotides.
Preferably, the modified nucleotide comprises a base selected from the group of bases consisting of guanine, adenine, uracil, thymine and cytosine having a functional group capable of receiving an electron from said base upon the modified nucleotide receiving photon energy. And, preferably, the functional group is photo-reactive.
A preferred functional group is selected from the group consisting of anthraquinone, naphthalene, phenanthrene, pterins, pyrene, quinidine, quinolone and derivatives thereof. Particularly preferred modified nucleoside are anthraquinoneacetyl-N(C6)-2′-deoxyadenine and N6-(anthraquinone-2-carbonyl)-2′-deoxyadenosine.
Preferably, the first nucleic acid is deoxyribose nucleic acid (DNA) and the signal nucleic acid is selected from the group of nucleic acids consisting of RNA replicase template nucleic acid, ribosomal RNA, messenger RNA, and target DNA and RNA nucleic acid.
Preferably, the photon source is pulsed and said charge monitor measures the difference between said first and second periods. The charge monitor is preferably phase locked with the photon source. This allows signal averaging at kilohertz or greater frequencies. A preferred photon source is a YAG laser or xenon flash light. The difference in the first and second periods can be detected as current through conventional equipment.
The electrode is preferably paired with a opposing electrode and the charge monitor measures current flowing between the electrodes as the electrodes are immersed in a sample potentially containing the signal nucleic acid.
A further embodiment of the present invention is a method for detecting the presence or absence of a signal nucleic acid. The method comprises the steps of providing a device having an electrode and a first nucleic acid covalently bound to the electrode acid to receive and donate electrons. The first nucleic acid has two or more donor nucleotides capable of donating an electron. The donor nucleotides have a position in the first nucleic acid where one donor nucleotide is proximal to the electrode. The first nucleic acid has a modified nucleoside adjacent to one of the donor nucleotides. The modified nucleotide is capable of receiving an electron from the donor nucleotides upon photo-excitation and maintaining the electron for a first period of time when the first nucleic acid is unhybridized and a second period of time when the first nucleic acid is hybridized to the signal nucleic acid. The first and second periods are different. The device further has a photon source for emitting photons on the first nucleic acid and, a charge monitor in communication with the electrode. The charge monitor measure the charge on the electrode or current flowing through the electrode as the first nucleic acid receives photons from the photon source. The charge on the electrode or current through the electrode is different in the presence of signal nucleic acid. The difference is indicative of the presence or absence of the signal nucleic acid. The method further comprises the step of contacting the first nucleic acid with a sample potentially containing the signal nucleic acid under conditions in which the signal nucleic acid will hybridize with the first nucleic acid. And, the method includes the step of generating photons f
Brusca John S.
Georgia University Research Foundation Inc.
Janiuk Anthony J.
Lundgren Jeffrey S.
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