Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry.

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.

Conformation matters: siRNAs with antisense strands with 5'-()-vinyl-phosphonate-α-L-LNA elicit stronger RNAi-mediated gene silencing than those with 5'-()-vinyl-phosphonate-LNA.

Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, ()-vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3'- (south) conformation, than when the antisense strand was modified with 5'-VP-LNA, which adopts a C3'- (north) pucker.

Recognition of mixed-sequence double-stranded DNA regions using chimeric Invader/LNA probes.

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, , double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2'--(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, , heteroduplex probes between individual Invader strands and complementary γPNA strands.

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.

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 hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), , single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with ()-hydroxymethyl moieties, (ii) Invader probes, , DNA duplexes modified with +1 interstrand zippers of 2'--(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, , 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.

Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA.

Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, , short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition.

Recognition of double-stranded DNA using LNA-modified toehold Invader probes.

Development of molecules capable of binding to specific sequences of double-stranded (ds) DNA continues to attract considerable interest, as this may yield useful tools for applications in life science, biotechnology, and medicine. We have previously demonstrated sequence-unrestricted of dsDNA using Invader probes, , DNA duplexes that are energetically activated through incorporation of +1 interstrand zipper arrangements of O2'-intercalator-functionalized RNA monomers. Nonetheless, recognition of extended dsDNA target regions remains challenging due to the high stability of the corresponding probes.

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.

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.

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.

Head-to-head comparison of LNA, γPNA, 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.

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.

Synthesis and biophysical characterization of oligonucleotides modified with O2'-alkylated RNA monomers featuring substituted pyrene moieties.

Over the past three decades, a wide range of pyrene-functionalized oligonucleotides have been developed and explored for potential applications in material science and nucleic acid diagnostics. Our efforts have focused on their possible use as components of Invader probes, i.e.

25 years and still going strong: 2'-O-(pyren-1-yl)methylribonucleotides - versatile building blocks for applications in molecular biology, diagnostics and materials science.

Oligonucleotides (ONs) modified with 2'-O-(pyren-1-yl)methylribonucleotides have been explored for a range of applications in molecular biology, nucleic acid diagnostics, and materials science for more than 25 years. The first part of this review provides an overview of synthetic strategies toward 2'-O-(pyren-1-yl)methylribonucleotides and is followed by a summary of biophysical properties of nucleic acid duplexes modified with these building blocks. Insights from structural studies are then presented to rationalize the reported properties.

Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2'-triphenylene- and coronene-functionalized RNA monomers.

Development of hybridization-based probes that enable recognition of specific mixed-sequence double-stranded DNA (dsDNA) regions is of considerable interest due to their potential applications in molecular biology, biotechnology, and medicine. We have recently demonstrated that nucleic acid duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl RNA monomers are inherently activated for recognition of mixed-sequence dsDNA targets, including chromosomal DNA. In the present work, we follow up on our previous structure-activity relationship studies and explore if the dsDNA-recognition efficiency of these so-called Invader probes can be improved by using larger intercalators than pyrene.

Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and--more recently--engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction.

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5-Pyrene-Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers.

Oligodeoxyribonucleotides modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2'-deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non-stringent conditions, relative to the corresponding LNA-free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore-labeled probes for SNP-discrimination studies.

Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes.

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding.

Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling.

Porphyrins were attached to LNA uridine building blocks via rigid 5-acetylene or more flexible propargyl-amide linkers and incorporated into DNA strands. The systems show a greatly increased thermodynamic stability when using as little as three porphyrins in a zipper arrangement. Thermodynamic analysis reveals clustering of the strands into more ordered duplexes with both greater negative ΔΔS and ΔΔH values, and less ordered duplexes with small positive ΔΔS differences, depending on the combination of linkers used.

Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles.

Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.

Synthesis and characterization of oligodeoxyribonucleotides modified with 2'-thio-2'-deoxy-2'-S-(pyren-1-yl)methyluridine.

Pyrene-functionalized oligonucleotides are intensively explored for applications in materials science and diagnostics. Here, we describe a short synthetic route to 2'-S-(pyren-1-yl)methyl-2'-thiouridine monomer S, its incorporation into oligodeoxyribonucleotides (ONs), and biophysical characterization thereof. Pseudorotational analysis reveals that the furanose ring of this monomer has a slight preference for South-type conformations.

Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers.

Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine and 2'-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization.

Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers.

LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides.

Recognition of Double-Stranded DNA Using Energetically Activated Duplexes Modified with N2'-Pyrene-, Perylene-, or Coronene-Functionalized 2'-N-Methyl-2'-amino-DNA Monomers.

Invader probes have been proposed as alternatives to polyamides, triplex-forming oligonucleotides, and peptide nucleic acids for recognition of chromosomal DNA targets. These double-stranded probes are activated for DNA recognition by +1 interstrand zippers of pyrene-functionalized nucleotides. This particular motif forces the intercalating pyrene moieties into the same region, resulting in perturbation and destabilization of the probe duplex.