Bright
signal outputs are needed for fluorescence detection of
biomolecules at their native expression levels. Increasing the number
of labels on a probe often results in crowding-induced self-quenching
of chromophores, and maintaining the function of the targeting moiety
(e.g., an antibody) is a concern. Here we demonstrate a simple method
to accommodate thousands of fluorescent dye molecules on a single
antibody probe while avoiding the negative effects of self-quenching.
We use a bottlebrush polymer from which extend hundreds of duplex
DNA strands that can accommodate hundreds of covalently attached and/or
thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA
assembly sequesters the intercalated fluorophores against dissociation
and can be tethered through DNA hybridization to an IgG antibody.
The resulting fluorescent nanotag can detect protein targets in flow
cytometry, confocal fluorescence microscopy, and dot blots with an
exceptionally bright signal that compares favorably to commercially
available antibodies labeled with organic dyes or quantum dots.
We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (Tm) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the Tm of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a Tm around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.
This review describes the application of peptide nucleic acids (PNAs) as clamps that prevent nucleic acid amplification of wild-type DNA so that DNA with mutations may be observed. These methods are useful to detect single-nucleotide polymorphisms (SNPs) in cases where there is a small amount of mutated DNA relative to the amount of normal (unmutated/wild-type) DNA. Detecting SNPs arising from mutated DNA can be useful to diagnose various genetic diseases, and is especially important in cancer diagnostics for early detection, proper diagnosis, and monitoring of disease progression. Most examples use PNA clamps to inhibit PCR amplification of wild-type DNA to identify the presence of mutated DNA associated with various types of cancer.
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