Analogs of dUTP and UTP that contain a biotin molecule covalently bound to the C-5 position of the pyrimidine ring through an allylamine linker arm have been synthesized. These biotin-labeled nucleotides are efficient substrates for a variety of DNA and RNA polynerases in vitro. Polynucleotides containing low levels ofbiotin substitution (50'molecules or fewer per kilobase) have denaturation, reassociation, and hybridization characteristics similar. to those of unsubstituted controls. Biotinlabeled polynucleotides, both single and double stranded, are selectively and quantitatively retained on avidin-Sepharose, even after extensive washing with 8 M urea, 6 M guanidine hydrochloride, or 99% formamide. In addition, biotin-labeled polynucleotides can be selectively immunoprecipitated in the presence of antibiotin antibody and Staphylococcus aureus protein A. The unique features ofbiotin-labeled polynucleotides suggest that they will be useful affinity probes for the detection and isolation of specific DNA and RNA sequences.Nucleotide analogs that can function as indicator "probes" when incorporated in polynucleotides would be of significant utility in many procedures used in biomedical and recombinant DNA research. When used in conjunction with immunological, histochemical, or affinity detector systems, such reagents could provide suitable alternatives to radioisotopes for the detection, localization, and isolation of nucleic acid components. Biotin (vitamin H) has many features that make it an ideal probe candidate. The interaction between biotin and avidin, a 68,000-dalton glycoprotein from egg white, has one ofthe highest binding constants (KY., = 10'5) known. (1). When avidin is coupled to appropriate indicator molecules (fluorescent dyes, electrondense proteins, enzymes, or antibodies), minute quantities of biotin can be detected (24). The specificity and tenacity of the biotin-avidin complex has been exploited to develop methods for the visual localization of specific proteins, lipids, and carbohydrates on or within cells (for review, see. ref. 2). Davidson and associates (9-11) chemically crosslinked biotin to RNA, via cytochrome c or polyamine bridges, and used these RNA-biotin complexes as probes for in situ hybridization. The sites of hybridization were visualized in the electron microscope through the binding of avidin-ferritin or avidin-methacrylate spheres. Although this approach to the detection of polynucleotide sequences was successful in the specialized cases examined, a simpler and more general procedure for preparing biotin-substituted nucleic acids was desirable. Biotin directly attached to a nucleotide that functions as an efficient polymerase substrate would be more versatile, both in the experimental protocols and in the detection methods that could be used.We have synthesized a number of nucleotide analogs that contain potential probe determinants (e.g., biotin, iminobiotin, and 2,4-dinitrophenyl groups) covalently attached to the pyrimidine or purine ring in the hope that one of-t...

The chemiluminescent reaction of an acridinium ester (AE) requires addition of peroxide to the 9 position of the acridinium ring. The addition of a hydroxide ion to the 9 position of an acridinium ester to form the carbinol adduct has also been well documented. We have observed a similar addition of other nucleophiles to the acridinium ring to form an acridan adduct. The adduct formed with bisulphite has been particularly well-characterized for rate of formation, rate of reversion, and reaction equilibrium. The formation of an adduct (other than H2O2) has been demonstrated to decrease significantly the reactivity of the adjacent ester bond to alkaline hydrolysis. The resulting, more stable adduct is very useful when the acridinium ester is used as a label in DNA probe-based assays. The adduct is highly resistant to hydrolysis under the conditions often desired for DNA probe-based assays (high temperature, elevated pH, extended storage).

Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.

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