Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes

Skoblov, Aleksander Yu.; Vichuzhanin, M. V.; Farzan, Valentina M.; Veselova, Olga A.; Konovalova, T. A.; Podkolzin, Alexander T.; Shipulin, German A.; Zatsepin, Timofei S.

doi:10.1016/j.bmc.2015.08.041

Cited by 7 publications

(9 citation statements)

References 53 publications

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“…We used a novel SIMA dye for the HEX channel as it proved to be more robust during oligonucleotide synthesis, deprotection and purification in comparison to the common HEX dye. 21 The R6G dye, 16 another alternative to HEX, increased the melting temperature of MB and MB duplexes with the target, thus complicating the design of MB pairs (data not shown). Therefore SIMA was found to be the optimal choice for MB synthesis and application for SNP detection.…”

Section: Resultsmentioning

confidence: 99%

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Specificity of SNP detection with molecular beacons is improved by stem and loop separation with spacers

Farzan

Markelov²,

Skoblov

et al. 2017

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Molecular beacons (MBs) are valuable tools in molecular biology, clinical diagnostics and analytical chemistry. Here we describe a novel approach for the design of MBs with nucleotide or non-nucleotide linkers between the stem and loop regions. Such modified MBs have significantly improved specificity and performance for single nucleotide polymorphism (SNP) detection. These advantages are especially distinct, when compared to the classic MBs, in the case of possible interactions between the stem and loop regions. We demonstrated the applicability of such modified MBs for the discrimination of common Factor V, NOS3 and ADRB2 SNPs in model plasmids and in clinical samples. The developed approach could be applicable not only to fluorescently labeled MBs, but also to other biosensors based on nucleic acids with stem-loop structures.

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Section: Resultsmentioning

confidence: 99%

“…The conditions of PAGE, HPLC purifications and analysis were the same as previously published. 16 ESI-MS spectra for oligonucleotides were recorded using a Bruker Maxis Impact q-TOF system as described earlier (Table S2 †).…”

Section: Oligonucleotide Synthesis Purification and Characterizationmentioning

confidence: 99%

Specificity of SNP detection with molecular beacons is improved by stem and loop separation with spacers

Farzan

Markelov²,

Skoblov

et al. 2017

Analyst

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“…The synthesis of azide, alkyne, and phosphoramidite derivatives (Figure ) is presented in the Supporting Information. HPLC analysis and the purification of oligonucleotides was carried out as described previously . Electrospray ionization mass spectrometry (ESI-MS) analysis for the oligonucleotides was performed using an Agilent 1260-Bruker Maxis Impact system as described earlier with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters

et al. 2017

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We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt) as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

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“…ESI buffer A: 10 mM diisopropylamine, 15 mM 1,1,1,3,3,3hexafluoroisopropanol in water; ESI buffer B: 10 mM diisopropylamine, 15 mM 1,1,1,3,3,3-hexafluoroisopropanol, 80% acetonitrile. ESI-MS analysis was performed according to the procedure described previously with minor changes [38]. Salts from samples were washed out with ESI buffer A (4 column volumes) followed by a step of 100% B (2 column volumes) with a flow rate of 0.3 mL/min; the temperature was 45°C.…”

Section: Oligonucleotide Characterizationmentioning

confidence: 99%

Pyrene-Modified DNA Aptamers with High Affinity to Wild-Type EGFR and EGFRvIII

Zavyalova

Турашев²,

Novoseltseva

et al. 2020

Nucleic Acid Therapeutics

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Nucleic acid aptamers have been proven to be a useful tool in many applications. Particularly, aptamers to epidermal growth factor receptor (EGFR) have been successfully used for the recognition of EGFR-expressing cells, the inhibition of EGFR-dependent pathways, and targeted drug delivery into EGFR-positive cells. Several aptamers are able to discriminate wild-type EGFR from its mutant form, EGFRvIII. Aptamers to EGFR have hairpin-like secondary structures with several possible folding variations. Here, an aptamer, previously selected to EGFRvIII, was chosen as a lead compound for extensive post-SELEX maturation. The aptamer was 1.5-fold truncated, the ends of the hairpin stem were appended with GC-pairs to increase thermal stability, and single pyrene modification was introduced into the aptamer to increase affinity to the target protein. Pyrene modification was selected from extensive computer docking studies of a library of thousands of chemicals to EGFR near the EGF-binding interface. The resulting aptamers bound extracellular domains of both variants of EGFR: EGFRwt and EGFRvIII with subnanomolar apparent dissociation constants. Compared with the initial aptamer, affinity to EGFRwt was increased up to 7.5-fold, whereas affinity to EGFRvIII was increased up to 4-fold.

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Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes

Cited by 7 publications

References 53 publications

Specificity of SNP detection with molecular beacons is improved by stem and loop separation with spacers

Specificity of SNP detection with molecular beacons is improved by stem and loop separation with spacers

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters

Pyrene-Modified DNA Aptamers with High Affinity to Wild-Type EGFR and EGFRvIII

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