Synthesis and biophysical characterization of oligonucleotides modified with O2'-alkylated RNA monomers featuring substituted pyrene moieties.

Over the past three decades, a wide range of pyrene-functionalized oligonucleotides have been developed and explored for potential applications in material science and nucleic acid diagnostics. Our efforts have focused on their possible use as components of Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. We have previously demonstrated that Invader probes based on 2'-O-(pyren-1-yl)methyl-RNA monomers are energetically activated for sequence-unrestricted recognition of chromosomal DNA targets under non-denaturing conditions. As part of ongoing efforts towards delineating structure-property relationships and optimizing Invader probes, we report the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2'-O-(7-neo-pentylpyren-1-yl)methyl-uridine monomer V and 2'-O-(7-tert-butyl-1-methoxypyren-5-yl)methyl-uridine monomer Y. ONs modified with monomer V display increased DNA affinity (ΔTm up to +10.5 °C), while Y-modified ONs display lower DNA affinity and up to 22-fold increases in fluorescence emission upon RNA binding. Although these monomers display limited potential as building blocks for Invader probes, their photophysical properties render them of interest for diagnostic RNA-targeting applications.

Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2'-triphenylene- and coronene-functionalized RNA monomers.

Development of hybridization-based probes that enable recognition of specific mixed-sequence double-stranded DNA (dsDNA) regions is of considerable interest due to their potential applications in molecular biology, biotechnology, and medicine. We have recently demonstrated that nucleic acid duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl RNA monomers are inherently activated for recognition of mixed-sequence dsDNA targets, including chromosomal DNA. In the present work, we follow up on our previous structure-activity relationship studies and explore if the dsDNA-recognition efficiency of these so-called Invader probes can be improved by using larger intercalators than pyrene. Oligodeoxyribonucleotides modified with 2'-O-(triphenylen-2-yl)methyl-uridine monomer X and 2'-O-(coronen-1-yl)methyl-uridine monomer Z form extraordinarily stabilized duplexes with complementary DNA (ΔTm's per modification of up to 13 °C and 20 °C, respectively). Invader probes based on X- and Z-monomers are shown to recognize model dsDNA targets with exceptional binding specificity, but are less efficient than reference probes modified with 2'-O-(pyren-1-yl)methyl-uridine monomer Y. The insight from this study will inform further optimization of Invader probes.

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.