Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA.

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.

PNA Clamping in Nucleic Acid Amplification Protocols to Detect Single Nucleotide Mutations Related to Cancer.

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.

Accessibility of Densely Localized DNA on Soft Polymer Nanoparticles.

The dense localization of DNA on soluble nanoparticles can lead to effects distinct from equivalent amounts of the DNA in solution. However, the specific effect may depend on the nature of the assembly and the nanoparticle core. Here we examine the accessibility of densely packed DNA duplexes that extend from a bottle-brush polymer core. We find that unlike spherical nucleic acids, the DNA duplex bristles on the bottle-brush polymer remain accessible to sequence-specific cleavage by endonucleases. In addition, the hybridized strand of the duplex can be displaced through a toehold-mediated strand exchange even at the polymer interface. These results demonstrate that the DNA on bottle-brush polymer remains sufficiently flexible to allow enzymatic degradation or DNA hybridization.

Pseudo-Ligandless Click Chemistry for Oligonucleotide Conjugation.

Particularly for its use in bioconjugations, the copper-catalyzed (or copper-promoted) azide-alkyne cycloaddition (CuAAC) reaction or 'click chemistry', has become an essential component of the modern chemical biologist's toolbox. Click chemistry has been applied to DNA, and more recently, RNA conjugations, and the protocols presented here can be used for either. The reaction can be carried out in aqueous buffer, and uses acetonitrile as a minor co-solvent that serves as a ligand to stabilize the copper. The method also includes details on the analysis of the reaction product. © 2016 by John Wiley & Sons, Inc.

Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles.

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.