Phosphorothioate Nucleotides and Oligonucleotides – Recent Progress in Synthesis and Application

Guga, Piotr; Koziołkiewicz, Maria

doi:10.1002/cbdv.201100130

Cited by 69 publications

(55 citation statements)

References 204 publications

(171 reference statements)

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“…The detection of microorganisms by FISH has significantly changed with the introduction of several types of synthetic oligonucleotides that possess a higher capacity of discrimination and affinity towards complementary sequences (Buchardt et al 1993;Obika et al 1997;Koshkin et al 1998;Majlessi et al 1998;Guimaraes et al 2007;Guga and Koziolkiewicz 2011). Therefore, it is very important to discuss which type of nucleic acid mimics is more suitable for each specific situation, such as the recognition of mismatches.…”

Section: Discussionmentioning

confidence: 99%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

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Section: Discussionmentioning

confidence: 99%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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“…1a). These species represented partially desulfurized oligonucleotides, which are commonly observed impurities of synthetic phosporothioate oligonucleotides (32,36). The other batch hardly contained any desulfurization product (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…They are usually produced with a modified phosphoramidite method of solid-phase synthesis (31,32). During every cycle of synthesis the oxidation of the internucleotide O-Me phosphite is replaced by sulfurization.…”

Section: Resultsmentioning

confidence: 99%

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Erb

Leithner²,

Bernkop‐Schnürch³

et al. 2012

AAPS J

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Abstract. Phosporothioate oligonucleotides represent an important class of therapeutic oligonucleotides, in which none-bridging oxygen atoms of the phosphate groups are replaced by sulfur. These oligonucleotides are designed to treat disease by modulating gene expression of an affected individual. As the development and application of these therapeutical oligonucleotides require analytical support, the development, validation, and application of an assay for the quantitative analysis of a phosporothioate oligonucleotide in rat plasma is described. The method employs ion-pair reversedphase chromatography on a monolithic capillary column with acetonitrile gradients in cyclohexyldimethylammonium acetate for separation and high-resolution tandem mass spectrometry for detection of nucleic acids. Chromatographic parameters (i.e. column temperature, mobile phase composition) as well as mass spectrometric parameters (i.e. spray voltage, gas flow, and capillary position, scan mode) have been optimized for sensitive oligonucleotide quantification. Furthermore, a solid-phase extraction method was developed which enabled processing of 10 μl of plasma. The five-point calibration curve showed linearity over the range of concentrations from 100 to 1,000 nM of the oligonucleotide. The limit of detection was 50 nM. The intra-and inter-day precision and accuracies were always better than 10.2 %. Using this assay, we performed a pharmacokinetic study of the phosporothioate oligonucleotide in rat treated with a single intravenous dose of 0.39 μmol/kg. The assay sensitivity was sufficient to study the early phase elimination of the oligonucleotide. Small amounts of the oligonucleotide were detectable up to 3 h after dosing.

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“…Both modifications lead to a slightly longer P-X bond as compared to the native P-O bond. The O-to-S substitution results in a higher preference for soft divalent metal cations, whereas the O-to-BH 3 substitution generally decreases affinity to metal cations and reduces H-bond acceptor properties (Stivers and Nagarajan 2006;Li et al 2007;Guga and Koziolkiewicz 2011). The O-to-F substitution at the terminal phosphate results in a smaller net negative charge and impairs H-bonding (Baranowski et al 2015).…”

Section: Resultsmentioning

confidence: 99%

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

Ziemniak

Mugridge

Kowalska

et al. 2016

RNA

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Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5 ′ m 7 G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m 7 GpppN) and m 7 GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m 7 G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m 7 Gp S ppp S m 7 G, inhibited Dcp1/2 20 times more efficiently than m 7 GpppN or m 7 GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m 7 Gp S ppp S m 7 G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m 7 Gp S ppp S m 7 G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.

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Phosphorothioate Nucleotides and Oligonucleotides – Recent Progress in Synthesis and Application

Cited by 69 publications

References 204 publications

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex

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