CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

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

Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence in situ hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), i.e., single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with (R)-hydroxymethyl moieties, (ii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of 2'-O-(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, i.e., duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.

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.

Chimeric γPNA-Invader probes: using intercalator-functionalized oligonucleotides to enhance the DNA-targeting properties of γPNA.

Gamma peptide nucleic acids (γPNAs), i.e., single-stranded PNA strands that are modified at the γ-position with (R)-diethylene glycol, and Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers, are two types of nucleic acid mimics that are showing promise for sequence-unrestricted recognition of double-stranded (ds) DNA targets. We recently demonstrated that recognition of dsDNA targets with self-complementary regions is challenging for single-stranded high-affinity probes like γPNAs due to their proclivity for secondary structure formation, but not so for Invader probes, which are engineered to form readily denaturing duplexes irrespective of the target sequence context. In the present study, we describe an approach that mitigates these limitations and improves the dsDNA-recognition properties of γPNAs in partially self-complementary target contexts. Chimeric probes between γPNAs and individual Invader strands are shown to form metastable duplexes that (i) are energetically activated for recognition of complementary mixed-sequence dsDNA target regions, (ii) reduce γPNA dimerization, and (iii) substantially improve the fidelity of the dsDNA-recognition process. Chimeric γPNA-Invader probes are characterized with respect to thermal denaturation properties, thermodynamic parameters associated with duplex formation, UV-Vis and fluorescence trends to establish pyrene binding modes, and dsDNA-recognition properties using DNA hairpin model targets.

Impact of non-nucleotidic bulges on recognition of mixed-sequence dsDNA by pyrene-functionalized Invader probes.

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.

Recognition of mixed-sequence DNA targets using spermine-modified Invader probes.

Double-stranded oligodeoxyribonucleotides with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers are additionally activated for highly specific recognition of mixed-sequence DNA targets upon incorporation of non-nucleotidic spermine bulges.

Head-to-head comparison of LNA, MPγPNA, INA and Invader probes targeting mixed-sequence double-stranded DNA.

Four probe chemistries are characterized and compared with respect to thermal denaturation temperatures (Tms), thermodynamic parameters associated with duplex formation, and recognition of mixed-sequence double-stranded (ds) DNA targets: (i) oligodeoxyribonucleotides (ONs) modified with Locked Nucleic Acid (LNA) monomers, (ii) MPγPNAs, i.e., single-stranded peptide nucleic acid (PNA) probes that are functionalized at the γ-position with (R)-diethylene glycol (mini-PEG, MP) moieties, (iii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers, and (iv) intercalating nucleic acids (INAs), i.e., DNA duplexes with opposing insertions of 1-O-(1-pyrenylmethyl)glycerol bulges. Invader and INA probes, which are designed to violate the nearest-neighbor exclusion principle, denature readily, whereas the individual probe strands display exceptionally high affinity towards complementary DNA (cDNA) as indicated by increases in Tms of up to 8 °C per modification. Optimized Invader and INA probes enable efficient and highly specific recognition of mixed-sequence dsDNA targets with self-complementary regions (C50 = 30-50 nM), whereas recognition is less efficient with LNA-modified ONs and fully modified MPγPNAs due to lower cDNA affinity (LNA) and a proclivity for dimerization (LNA and MPγPNA). A Cy3-labeled Invader probe is shown to stain telomeric DNA of individual chromosomes in metaphasic spreads under non-denaturing conditions with excellent specificity.