bc9b00746_si_001.pdf (1.58 MB)
Fluorescent Oligonucleotides with Bis(prop-2-yn-1-yloxy)butane-1,3-diol Scaffold Rapidly Detect Disease-Associated Nucleic Acids
journal contribution
posted on 2019-12-04, 16:39 authored by Maria Taskova, Kira AstakhovaBiomedical research and clinical work demand rapid and
reliable
detection of disease-associated nucleic acids. Fluorescent oligonucleotides
that bind precisely, and sense target DNA or RNA, are useful tools
for simple hybridization-based assays. Although a plethora of oligonucleotide
modifications are reported in the literature, they often result in
poor coupling yields and are very expensive. We describe the synthesis
of a new bisalkyne butane-1,3-diol scaffold and its efficient coupling
into oligonucleotide sequences. We hypothesized that covalent attachment
of multiple (2/4) fluorescent groups to the scaffold within oncogene-specific
oligonucleotides could lead to beneficial detection of target DNA.
To test this, we post-synthetically conjugated the oligonucleotides
with azide-derivative dyes (2/4 per sequence): perylene, 5JOE, and
(phenylethynyl)pyrene. We investigated the biophysical and photophysical
properties of the oligonucleotide–dye conjugates and confirmed
a “light up” fluorescent sensing mechanism of the probes
upon target binding. However, fluorescence of the probes was not sensitive
to mismatches. Nevertheless, “clicked” probes showed
a high specificity of binding to complementary target, with the difference
in Tm over 10 °C for matched vs mismatched
duplex. When applied together, the mismatch-induced difference in
temperature melting and fluorescence-based discrimination of the target-bound
vs single-stranded probe state allowed us to apply the perylene conjugates
to detect mutations in human oncogenes. Due to the beneficial target
binding properties of the perylene labeled probes, along with the
high fluorescence intensity of probe:target duplexes, human oncogenes
could be detected in a convenient and fast (2.5 h) bead-based assay.
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sense target DNAoligonucleotide sequencescovalent attachmentbead-based assayfluorescence intensitytarget bindingfluorescence-based discriminationperylene conjugatesoligonucleotide modificationstarget DNARNAT mFluorescent Oligonucleotidesphotophysical propertiestarget binding propertiesFluorescent oligonucleotidestarget-bound vs single-stranded probe statework demandoncogene-specific oligonucleotideshybridization-based assaysmismatch-induced difference5 JOE
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