Patrick J. Hrdlicka

Mutation hotspots in melanoma frequently occur at DNA binding sites of E26 transformation-specific (ETS)-family transcription factors, as ETS factors stimulate the formation of UV-induced cyclobutane pyrimidine dimers (CPDs) while suppressing repair at ETS-bound DNA sites. To elucidate the molecular mechanism by which ETS factors bind to damaged DNA sites and inhibit repair, we investigated the binding of members from the three major classes of the ETS superfamily (Ets1, ELF1, and PU.1) to cognate DNA containing a cis-syn TpT CPD. These site-specific CPDs modulated ETS recognition and repair by a model repair enzyme in a position-dependent manner. Specifically, a deaminated CPD located in a damage hotspot in the ETS binding motif consistently stimulated binding and inhibited T4 PDG (a CPD repair enzyme) by all three paralogs. Co-crystal structures of PU.1 reveal that CPDs and mismatches are recognized within the framework of canonical ETS/DNA complexes. Molecular dynamics simulations in explicit solvent show that CPD introduces compensatory structural dynamics to both the free and ETS-bound states that strongly modify the underlying thermodynamics of recognition. The results offer a molecular basis for how ETS factors induce mutation hotspots in skin cancers and other UV-exposed tissues by binding to CPD-containing sites and inhibiting their repair.

Invader are double-stranded oligonucleotide probes with specific zippers of intercalator-functionalized nucleotides that enable efficient and highly specific recognition of complementary DNA regions via a strand-invasion mechanism.

A comprehensive and user-friendly method for evaluating recognition of double-stranded DNA (dsDNA) targets by oligonucleotide-based probes is presented. Thus, dsDNA-targeting probes such as single-stranded locked nucleic acids (LNAs) and double-stranded Invader probes are incubated with digoxigenin-labeled DNA hairpin targets, and the resulting recognition complexes are resolved using an electrophoretic mobility shift assay and tagged using a chemiluminescence immunoassay. Emissive products are detected by a C-DiGit Blot Scanner and quantified with the accompanying software. R-based scripts for data visualization and determination of C ₅₀ values (a measure of the dsDNA-binding affinity of a probe) are also provided. The data presented here demonstrate the effectiveness of the described protocol and highlight the variable dsDNA-recognition efficiencies of LNAs, Invader probes, and chimeric Invader:LNA probes.

Single-stranded LNA/MOE mixmers and duplexes between intercalator-functionalized oligonucleotides and Xeno RNAs efficiently recognize double-stranded DNA with exceptional binding affinity.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

RNAi-mediated gene silencing is more efficient when the antisense strand of the siRNA is modified at 5′ end with a 5′-vinyl-phosphonate carrying nucleotide that adopts a C3′-exo (south) conformation than a C3′-endo (north) pucker.

Development of robust oligonucleotide-based probe technologies, capable of recognizing specific regions of double-stranded DNA (dsDNA) targets, continues to attract considerable attention due to the promise of tools for modulation of gene expression, diagnostic agents, and new modalities against genetic diseases. Our laboratory pursues the development of various strand-invading probes. These include Invader probes, i.e., double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2′-O-(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, i.e., heteroduplex probes between individual Invader strands and complementary γPNA strands. Here we report on the biophysical properties and dsDNA-recognition characteristics of a new class of chimeric probes—chimeric Invader/LNA probes—which are comprised of densely modified Invader strands and fully modified complementary LNA strands. The chimeric Invader/LNA probes form labile and distorted heteroduplexes, due to an apparent incompatibility between intercalating pyrene moieties and LNA strands. In contrast, the individual Invader and LNA strands form very stable duplexes with complementary DNA, which provides the driving force for near-stoichiometric recognition of model double-stranded DNA targets with single base-pair accuracy. The distinctive properties of chimeric Invader/LNA probes unlock exciting possibilities in molecular biology, and diagnostic and therapeutic fields.

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.

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.