Open AccessImpact of non-nucleotidic bulges on recognition of mixed-sequence dsDNA by pyrene-functionalized Invader probes
Author(s) -
Dale C. Guenther,
Raymond G Emehiser,
Allison Inskeep,
Saswata Karmakar,
Patrick J. Hrdlicka
Publication year - 2020
Publication title -
organic and biomolecular chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 146
eISSN - 1477-0539
pISSN - 1477-0520
DOI - 10.1039/d0ob01052a
Subject(s) - pyrene , chemistry , sequence (biology) , dna , benzo(a)pyrene , evolutionary biology , computational biology , biology , biochemistry , organic chemistry
Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, are energetically activated for sequence-unrestricted recognition of double-stranded DNA (dsDNA) as they are engineered to violate the neighbor exclusion principle, while displaying high affinity towards complementary DNA sequences. The impact on Invader-mediated dsDNA-recognition upon additional modification with different non-nucleotidic bulges is studied herein, based on the hypothesis that bulge-containing Invader probes will display additionally disrupted base-stacking, more extensive denaturation, and improved dsDNA-recognition efficiency. Indeed, Invader probes featuring a single central large bulge - e.g., a nonyl (C9) monomer - display improved recognition of model DNA hairpin targets vis-à-vis conventional Invader probes (C50 values ∼1.5 μM vs. ∼3.9 μM). In contrast, probes with two opposing central bulges display less favorable binding characteristics. Remarkably, C9-modified Invader probes display perfect discrimination between fully complementary dsDNA and dsDNA differing in only one of eighteen base-pairs, underscoring the high binding specificity of double-stranded probes. Cy3-labeled bulge-containing Invader probes are demonstrated to signal the presence of gender-specific DNA sequences in fluorescent in situ hybridization assays (FISH) performed under non-denaturing conditions, highlighting one potential application of dsDNA-targeting Invader probes.