48 research outputs found
Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2-or 4-pyrenyl-functionalized O2 '-alkylated RNA monomers
Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics
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 ??S and ??H values, and less ordered duplexes with small positive ??S differences, depending on the combination of linkers used. The exciton coupling between the porphyrins is dependent on the flanking DNA sequence in the single stranded form, and on the nature of the linker between the nucleobase and the porphyrin in the double stranded form; it is, however, also strongly influenced by intermolecular interactions. This system is suitable for the formation of stable helical chromophore arrays with sequence and structure dependent exciton coupling
Polymerase-directed synthesis of C5-ethynyl locked nucleic acids
Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5′-triphosphate as substrate for RNA transcripts
Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes
Glycerol kinase plays a critical role in metabolism by converting glycerol to glycerol 3-phosphate in an ATP dependent reaction. In humans, glycerol kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperglycerolemia and glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for glycerol kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced glycerol kinase RNA expression, reduced phosphorylation activity and elevated glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with glycerol. Due to the hygroscopic nature of glycerol, we predict glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting glycerol kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of glycerol hypersensitivity. Furthermore, we found that the glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the glycerol hypersensitive phenotype identified in our Drosophila model for glycerol kinase deficiency
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. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms
C2′-Pyrene-Functionalized Triazole-Linked DNA: Universal DNA/RNA Hybridization Probes
Development of universal hybridization probes, that is,
oligonucleotides
displaying identical affinity toward matched and mismatched DNA/RNA
targets, has been a longstanding goal due to potential applications
as degenerate PCR primers and microarray probes. The classic approach
toward this end has been the use of “universal bases”
that either are based on hydrogen-bonding purine derivatives or aromatic
base analogues without hydrogen-bonding capabilities. However, development
of probes that result in truly universal hybridization without compromising
duplex thermostability has proven challenging. Here we have used the
“click reaction” to synthesize four C2′-pyrene-functionalized
triazole-linked 2′-deoxyuridine phosphoramidites. We demonstrate
that oligodeoxyribonucleotides modified with the corresponding monomers
display (a) minimally decreased thermal affinity toward DNA/RNA complements
relative to reference strands, (b) highly robust universal hybridization
characteristics (average differences in thermal denaturation temperatures
of matched vs mismatched duplexes involving monomer <b>W</b> are <1.7 °C), and (c) exceptional affinity toward DNA targets
containing abasic sites opposite of the modification site (Δ<i>T</i><sub>m</sub> up to +25 °C). The latter observation,
along with results from absorption and fluorescence spectroscopy,
suggests that the pyrene moiety is intercalating into the duplex whereby
the opposing nucleotide is pushed into an extrahelical position. These
properties render C2′-pyrene-functionalized triazole-linked
DNA as promising universal hybridization probes for applications in
nucleic acid chemistry and biotechnology
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
Synthesis, Hybridization Characteristics, and Fluorescence Properties of Oligonucleotides Modified with Nucleobase-Functionalized Locked Nucleic Acid Adenosine and Cytidine Monomers
Conformationally
restricted nucleotides such as locked nucleic
acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing
modifications in oligonucleotide chemistry to produce probes for nucleic
acid targeting applications in molecular biology, biotechnology, and
medicinal chemistry. Considerable efforts have been devoted in recent
years to optimize the biophysical properties of LNA through additional
modification of the sugar skeleton. We recently introduced C5-functionalization
of LNA uridines as an alternative and synthetically more straightforward
approach to improve the biophysical properties of LNA. In the present
work, we set out to test the generality of this concept by studying
the characteristics of oligonucleotides modified with four different
C5-functionalized LNA cytidine and C8-functionalized LNA adenosine
monomers. The results strongly suggest that C5-functionalization of
LNA pyrimidines is indeed a viable approach for improving the binding
affinity, target specificity, and/or enzymatic stability of LNA-modified
ONs, whereas C8-functionalization of LNA adenosines is detrimental
to binding affinity and specificity. These insights will impact the
future design of conformationally restricted nucleotides for nucleic
acid targeting applications