Several sequences forming G-quadruplex are highly conserved in regulatory regions of genomes of different organisms and affect various biological processes like gene expression. Diverse G-quadruplex properties can be modulated via their interaction with small polyaromatic molecules such as pyrene. To investigate how pyrene interacts with G-rich DNAs, we incorporated deoxyuridine nucleotide(s) with a covalently attached pyrene moiety (Upy) into a model system that forms parallel G-quadruplex structures. We individually substituted terminal positions and positions in the pentaloop of the c-kit2 sequence originating from the KIT proto-oncogene with Upy and performed a detailed NMR structural study accompanied with molecular dynamic simulations. Our results showed that incorporation into the pentaloop leads to structural polymorphism and in some cases also thermal destabilization. In contrast, terminal positions were found to cause a substantial thermodynamic stabilization while preserving topology of the parent c-kit2 G-quadruplex. Thermodynamic stabilization results from π-π stacking between the polyaromatic core of the pyrene moiety and guanine nucleotides of outer G-quartets. Thanks to the prevalent overall conformation, our structures mimic the G-quadruplex found in human KIT proto-oncogene and could potentially have antiproliferative effects on cancer cells.

• MeSH
• deoxyuridin chemie   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• promotorové oblasti (genetika) MeSH
• protoonkogen Mas MeSH
• protoonkogenní proteiny c-kit genetika   MeSH
• pyreny chemie   MeSH
• termodynamika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyuridin MeSH
• KIT protein, human MeSH Prohlížeč
• MAS1 protein, human MeSH Prohlížeč
• protoonkogen Mas MeSH
• protoonkogenní proteiny c-kit MeSH
• pyrene MeSH Prohlížeč
• pyreny MeSH

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.

• MeSH
• afinita protilátek MeSH
• barvení a značení MeSH
• biologický transport MeSH
• biotinylace MeSH
• bromodeoxyuridin chemie   imunologie   metabolismus   MeSH
• deoxyuridin analogy a deriváty   chemie   imunologie   metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie * MeSH
• HeLa buňky MeSH
• lidé MeSH
• protilátky chemie   imunologie   MeSH
• zkřížené reakce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5-ethynyl-2'-deoxyuridine MeSH Prohlížeč
• bromodeoxyuridin MeSH
• deoxyuridin MeSH
• DNA MeSH
• fluorescenční barviva MeSH
• protilátky MeSH