Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-10-10
2002-08-06
Whisenant, Ethan C. (Department: 1634)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C435S006120, C435S091100, C436S094000
Reexamination Certificate
active
06428667
ABSTRACT:
INTRODUCTION
1. Technical Field
The field of this invention is fluorescent compositions and methods employing fluorescent compositions.
2. Background
Detection of fluorescent signals finds wide applications in a variety of situations and under a variety of conditions. Fluorescence has many advantages as a means of generating a detectable signal. Fluorescence does not suffer from the many disadvantages of a radioactive label, while in many cases it provides for a high level of sensitivity. Instrumentation for detection of fluorescence is readily available and fluorescent labels have found application in such diverse situations as immunodiagnostics, detection of nucleic acid bands in gel electrophoresis and in fluorescence activated cell sorters. The sensitivity of the fluorescent signal depends upon a number of factors: the possibility of self quenching, the effect of other molecules associated with the fluorescent molecule on the quantum efficiency of the fluorescence, the effect of the medium on the quantum efficiency and fluorescence characteristics of the fluorescer; the stability of the fluorescer to light, the ability to remove background fluorescence, and the like.
Desirably, we would wish to have a fluorescent label which was stable, both chemically and to light, provided a high quantum efficiency, was relatively insensitive to interactions with a variety of molecules, as well as variations in medium, had high light absorption and emission characteristics, was relatively insensitive to self-quenching, and could be readily attached to a wide variety of molecules under varying conditions without adversely affecting the fluorescent characteristics.
Relevant Literature
The following references describe DNA intercalating fluorescent dimers and their physical characteristics: Gaugain et al., Biochemistry 17, 5071-5078, 1978; Gaugain et al., Biochemistry 17, 5078-5088, 1978; Markovits et al., Anal. Biochemistry 94, 259-269, 1979; Markovits Biochemistry 22, 3231-3237, 1983; and Markovits et al., Nucl. Acids Res. 13, 3773-3788, 1985. Interaction of various intercalating compounds with nucleic acids is reviewed by Berman and Young,
Ann. Rev. Biophys. Bioeng
. (1986) 10:87-224. Retention of ethidium bromide on electrophoresis of the dye with DNA or RNA is described by Angemuller and Sayavedra-Soto, Biotechniques 8, 36, 1990 and Rosen and villa-Komaroff, Focus 12, 23, 1990.
SUMMARY OF THE INVENTION
Methods and compositions are provided for detecting molecules using fluorescent labels, where fluorescent intercalating molecules having strong binding affinities for nucleic acids are employed. The nucleic acid acts as a scaffold for the fluorescent non-covalently binding and intercalating compounds, minimizing self-quenching and providing for high fluorescence efficiency. The fluorescent labeling finds use in electrophoresis, diagnostic assays, cell labeling, and the like.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel methods and compositions are provided employing nucleic acid intercalating moieties, having at least one, usually at least two, fluorescent monomeric units, where the monomeric units have affinity for dsDNA (double stranded DNA). In considering the subject compositions a nucleic acid monointercalator complex intends a complex with one dye molecule, whether the dye molecule has a single or plurality of fluorescent monomeric units. A dye nucleic acid aggregate is an assemblage of nucleic acid containing many non-covalently bound and intercalated dye molecules.
The nucleic acid may be single stranded (ss) usually having hairpins, or double stranded (ds), RNA, DNA or combinations thereof, particularly dsDNA.
The monomers will have high binding affinity to the nucleic acid, generally having two or more positive charges. The multimeric compounds will also have high binding affinity for nucleic acids, although fewer than all the fluorescent intercalating units may non-covalently bind and intercalate, e.g., only one unit of a dimer, where each unit will have at least one positive charge, usually at least about two positive charges per unit.
The intercalating compounds may be monomers or homo- or heteropolymers with an affinity for dsDNA of at least about 5×10
6
M
−1
, more usually at least about 10
7
and greater than about 10
9
M
−1
at ionic strengths of at least about 0.01 usually at least about 0.04, preferably at least about 0.2 at 25° C. Gel electrophoresis is usually performed at an ionic strength of about 0.04.
The compounds are further characterized by employing fluorescent monomeric units which are cyclic, polycyclic, particularly polycyclic aromatic having at least two rings, usually at least three rings, and not more than about six rings, more usually not more than five rings, where at least two of the rings are fused, usually at least three of the rings. The aromatic compound may be carbocyclic or heterocyclic, particularly having from one to three, more usually one to two nitrogen atoms, as annular atoms. The monomeric units will be joined by a linking chain which will normally be of a length to allow for simultaneous intercalation to adjacent monomeric units in dsDNA, usually providing a length of at least about ten Angstroms, usually having at least about 9 atoms, more usually at least about ten atoms in the chain, and usually not more than about 26, more usually not more than 20 atoms, between fluorescent units. The linking group will usually be aliphatic, having from 0 to 8, more usually from 0 to 6, preferably from about 2 to 6 heteroatoms in the chain, particularly heteroatoms which provide for a positive charge, e.g. nitrogen and sulfur. Preferably, there will be at least one positive charge, more preferably at least two positive charges, usually not more than about 8 positive charges, more usually not more than about 6 positive charges.
The rings may be substituted by a wide variety of substituents, which substituents may include alkyl groups of from 1 to 4 carbon atoms, usually from 1 to 2 carbon atoms, oxy, which includes hydroxy, alkoxy and carboxy, generally of from 1 to 4 carbon atoms, amino, including mono- and disubstituted amino, particularly mono- and dialkylamino, thio, particularly alkylthio of from 1 to 4, usually 1 to 2 carbon atoms, cyano, nonoxo-carbonyl, such as carboxy, particular carboxamide or carboalkoxy, of from 2 to 6, usually 2 to 4 carbon atoms, oxo-carbonyl or acyl, generally of from 1 to 4 carbon atoms, halo, particularly of atomic number 9 to 35, etc.
The polymers will have at least two monomeric units and usually not more than 12 monomeric units, more usually not more than about 8 monomeric units, preferably not more than about 4 monomeric units.
Polycyclic compounds which find use include phenanthridines, acridines, porphyrins, phenylindoles, and bisbenzimides. Derivatives of these compounds which find use include, bis-(3,8-diamino-6-hydroxy-6-phenyl-5,6-dihydrophenanthridine, di-(7-hydropyridocarbazoles), tetraacridinylamine, hexa-acridinylamine, thiazole orange dimer, 5-(11-(2-methoxy-6-chloro-9-aminoacridinyl)-(4,8-diazaundecyl)-3,8-diamino-6-phenylphenan-thridinium chloride, and the like.
Compounds can be prepared from alkylene polyamines, where the alkylene groups are of from 2-10, usually 2-6 carbon atoms, and haloalkyl- or pseudohaloalkyl substituted fluorescent polycyclic aromatic compounds, e.g., phenanthridines or acridines, which may be substituted or unsubstituted, to provide for ternary or quaternary amino groups. The amino groups may be quaternized with any convenient alkylation agent, either before or after reaction with the fluorescent compound or may be prepared initially as ternary amines using alkylamines, where the alkyl group will be of from about 1-6, usually 1-3 carbon atoms. Illustrative of a compound would be N, N, N′, N″, N″,-pentamethyl-N, N′, N″-tris-(3,8-diamino-6-hydroxy-6-phenyl-5,6-dihydrophenanthridine).
These compounds find use as labeling agents, where the compounds are used in a process for detection of nucleic acid or as a label which is prep
Glazer Alexander N.
Mathies Richard A.
Peck Konan
Bozicevic, Field & Francis
Field Bret E.
Lu Frank
The Regents of the University of California, Berkeley
Whisenant Ethan C.
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