Serine-γPNA, Invader probes, and chimeras thereof: three probe chemistries that enable sequence-unrestricted recognition of double-stranded DNA

Emehiser, Raymond G; Dhuri, Karishma; Shepard, Caroline P; Karmakar, Saswata; Bahal, Raman; Hrdlicka, Patrick J.

doi:10.1039/d2ob01567f

Cited by 1 publication

(6 citation statements)

References 55 publications

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“…Conditions are as described in Figure 3. Data previously shown in [23]-reproduced with permission from the Royal Society of Chemistry.…”

Section: Recognition Of Mixed-sequence Model Dna Hairpin Targetsmentioning

confidence: 99%

“…Thus, nuclei that were pre-treated with DNase I did not produce any signals (Figure S18), whereas pre-treatment with RNase A or Proteinase K continued to yield single punctate signals, albeit with lower intensity (Figure S19 Incubation conditions and the image capture process were as described in Figure 6. Data previously shown in [23]-reproduced with permission from the Royal Society of Chemistry.…”

Section: Targeting Chromosomal Dna-fluorescence In Situ Hybridization...mentioning

confidence: 99%

“…A key advantage of strand-invading strategies is the prospect of the sequenceunrestricted recognition of dsDNA. Progress towards this end has been realized with various modified single-stranded PNAs [7][8][9][10][11][12], double-stranded probes such as pseudocomplementary (pc) PNAs [13][14][15], and related approaches [16][17][18][19][20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

“…The difference in stability between the probe-target duplexes, vis-à-vis the double-stranded Invader probe and the dsDNA target region, generates the driving force for dsDNA recognition via double-duplex strand invasion (Figure 1) [24]. The sequence-unrestricted recognition of dsDNA targets using Invader probes has been demonstrated, enabling the detection of (i) DNA fragments from specific food pathogens using sandwich assays [32], (ii) telomeric DNA of individual chromosomes in metaphasic spreads [33], and (iii) sex chromosome-specific targets in interphase and metaphase nuclei under non-denaturing conditions [23,24].…”

Section: Introductionmentioning

confidence: 99%

“…Note the absence of Cy3 signals.Incubation conditions and the image capture process were as described in Figure6. Data previously shown in[23]-reproduced with permission from the Royal Society of Chemistry.…”

mentioning

confidence: 99%

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Factors Impacting Invader-Mediated Recognition of Double-Stranded DNA

et al. 2022

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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.

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“…Conditions are as described in Figure 3. Data previously shown in [23]-reproduced with permission from the Royal Society of Chemistry.…”

Section: Recognition Of Mixed-sequence Model Dna Hairpin Targetsmentioning

confidence: 99%

Section: Targeting Chromosomal Dna-fluorescence In Situ Hybridization...mentioning

confidence: 99%