Application of Cu(I)-catalyzed azide–alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA

Abstract

SummaryEfficient protocols based on Cu(I)-catalyzed azide–alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole–imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these conjugates did not affect the binding parameters (affinity and triplex stability) of the parent TFOs.

Optimizing Mannose “Click” Conjugation to Polymeric Nanoparticles for Targeted siRNA Delivery to Human and Murine Macrophages

“…MnPEGDB Is Complexed with Small Oligonucleotides (dsDNA or siRNA) for 30 min in a pH 4 Buffer a a The protonated amine group on the DMAEMA electrostatically interacts with the negative phosphates on the oligonucleotide, spontaneously forming a stable micelle with mannose presented on the corona of the polyplex. After fabrication of the MnPEGDB and removal of residual copper using a Chelex resin following previously published methods, 23,43 the polymers were dissolved in ethanol and molecular-grade water. Residual copper was measured using a colorimetric assay that detects Cu 2+ ions, and the assay results (copper ions in μg/dL) were normalized to provide mg of copper per mg of polymer.…”

“…After fabrication of the MnPEGDB and removal of residual copper using a Chelex resin following previously published methods, , the polymers were dissolved in ethanol and molecular-grade water. Residual copper was measured using a colorimetric assay that detects Cu 2+ ions, and the assay results (copper ions in μg/dL) were normalized to provide mg of copper per mg of polymer.…”

Raman Imaging of Individual Membrane Lipids and Deoxynucleoside Triphosphates in Living Neuronal Cells during Neurite Outgrowth

“…The following kits were used as primary antibodies for immunocytostaining: (i) cell nuclei stain Hoechst 33342 (1:100) (2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1 H -benzimidazole trihydrochloride trihydrate, commercialized by Dojindo, Kumamoto, Japan), (ii) mouse anti-OPPC (dilution 1:500) (Cosmo Bio Company, Tokyo, Japan), and (iii) EdU (5-ethynyl-2′-deoxyuridine; Molecular Probes, Invitrogen, Thermo Fisher Scientific, Tokyo, Japan), which was covalently incorporated into DNA as a mimic of thymidine in order to visualize the cell nucleus. The terminal alkyne group was reacted with fluorescent azide in the major groove of the double helix (Alexa568-azide under Cu­(I)-catalyzed under click reaction conditions). , Immunoassaying was visualized by means of confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan).…”

“…The terminal alkyne group was reacted with fluorescent azide in the major groove of the double helix (Alexa568-azide under Cu(I)-catalyzed under click reaction conditions). 5,42 Immunoassaying was visualized by means of confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan).…”

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…More recently, in order to improve the specificity and to facilitate cellular penetration, the high affine TINA-TFOs (antiparallel and parallel types) have been conjugated to pyrrole-imidazole polyamide minor groove binders (MGB). However, it was shown that the MGBs, as part of the conjugates, do not affect the binding properties of the parent TINA-TFOs [ 215 ].…”