Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′‑<i>O</i>‑(Pyren-1-yl)methyl-RNA Monomers

The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies <i>Invaders</i> as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA <b><u>A</u></b>/<b><u>C</u></b>/<b><u>G</u></b>/<b><u>U</u></b> monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising <b><u>C</u></b> and/or <b><u>U</u></b> monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology

New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH

The discovery of two new classes of catalysts for hydrazone and oxime formation in water at neutral pH, namely 2-aminophenols and 2-(amino­methyl)­benz­imidazoles, is reported. Kinetics studies in aqueous solutions at pH 7.4 revealed rate enhancements up to 7-fold greater than with classic aniline catalysis. 2-(Aminomethyl)­benzimidazoles were found to be effective catalysts with otherwise challenging aryl ketone substrates

Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with “+1 interstrand zippers” of 2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2′-amino-α-l-LNA, 2′-<i>N</i>-methyl-2′-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2′-<i>O</i>-(pyren-1-yl)­methyl-RNA or 2′-<i>N</i>-methyl-2′-<i>N</i>-(pyren-1-yl)­methyl-2′-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics