3 DNA dendrimer signal amplification
The name 'dendrimer', derived from the Greek word for 'tree', suggests the unusual structure of this highly branched molecule. As a class, dendrimers are complex, branched molecules built from interconnected natural or synthetic monomeric subunits. A 3DNA dendrimer is constructed from DNA monomers – as the name '3DNA' indicates. Each 3DNA monomer is composed of two DNA strands that share a region of sequence complementarity located in the central portion of each strand. When the two strands anneal to form the monomer the resulting structure has a central double-stranded 'waist' bordered by four single-stranded 'arms'. This 'waist' plus 'arms' structure comprises the basic 3DNA monomer.
The arborescent structure of dendritic molecules makes them extremely useful for the development of nucleic acid diagnostics as signal amplification tools. Further, due to the relatively large size of nucleic acid molecules, nucleic acid dendrimers could be readily labeled with numerous fluorescent compounds and/or protein moieties. DNA dendrimers provide a generic method for amplifying signal and have general utility in nucleic acid blot assays. Dendritic DNA molecules (3DNA) could be readily labeled with numerous fluorescent compounds. Specificity to various DNA sequences is conferred to the dendrimers by hybridizing and covalently crosslinking oligonucleotides to the single-stranded surface of the dendrimers. Genisphere Inc is currently investigating applications of the dendrimer in Southern, Northern, and Western blots, fluorescent in-situ hybridization (FISH), and flow fluorescence assays. In a hybridization reaction, signal intensity is determined by the amount of label that can be localized at the reaction site. The 3DNA dendrimers in all of Genisphere kits are labeled with an average of at least 200 labels (more in some kits). The dendrimer carries this number of labels with it every time it hybridizes to a complementary molecule. The result is up to a 200-fold passive enhancement of signal intensity.
A versatile and strong signal amplification method is based on activities of a DNA polymerase to generate high concentrations of pyrophosphate (PPi), which is catalyzed by nucleotide extension and excision activities of a DNA polymerase on an oligonucleotide cassette. The signal is generated upon enzymatic conversion of PPi to ATP and ATP levels subsequently detected with firefly luciferase. Bioluminescence produced by an oligonucleotide cassette consisting of just two polymerase reaction sites is sufficient to detect them at low attomole levels. The attachment of a large number of these oligonucleotide cassettes to DNA dendrimers enables the detection of such polyvalent substrate molecules at low zeptomole concentrations. The extent of signal amplification obtained with dendrimer substrates is comparable to exponential target amplifications provided by nucleic acid amplification methods. The attachment of such PPi-generating dendritic DNA platforms to ligands that mediate target recognition would potentially permit detection of extremely low concentrations of analytes in diagnostic assays.
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