17 research outputs found
Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers
Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics
Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes
Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms
Factors Impacting Invader-Mediated Recognition of Double-Stranded DNA
The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman’s rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences
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)Âmethyl-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)Âmethyl-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)Âmethyl-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-(aminoÂmethyl)ÂbenzÂimidazoles, 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)Âbenzimidazoles
were found to be effective catalysts with otherwise challenging aryl
ketone substrates