20.500.12556/RUL-186327 Karakterizacija G-kvadrupleksov z dolgimi zankami v promotorju gena BCL-2 Characterization of long-looped G-quadruplexes in the BCL-2 promoter DNA je izredno dinamičen nosilec genetske informacije, ki se poleg dvojne vijačnice lahko zvije tudi v širok nabor nekanoničnih struktur. Med temi so najbolj znani Gkvadrupleksi, ki se uveljavljajo kot pomembni regulatorji izražanja genov. V preteklosti so se študije G-kvadrupleksov osredotočale predvsem na motive, ki sledijo ustaljenemu konsenznemu zaporedju, danes pa se področje širi tudi na alternativne oblike Gkvadrupleksov, izmed katerih so ti z dolgimi zankami še posebej zanimivi s stališčaspecifičnih interakcij s proteini in malimi molekulami – ligandi. V promotorju gena BCL-2, ki je znan po pomembnih z gvanini bogatih regijah, smo identificirali več potencialnih G-kvadrupleksov z dolgimi zankami, ki se zaradi slabo izbranih motivov niso zvili v želene strukture. S premišljeno modifikacijo zaporedij smo uspeli določiti ključne karakteristike, ki omogočajo tvorbo tovrstnih struktur. Najpomembnejšo vlogo imajo G-trakti, ki ne smejo vsebovati izboklin, pri daljših zankah pa k zvijanju močno prispevajo tudi Watson-Crickovi bazni pari. Z optimizacijo zaporedij smo prišli do modelnih vzorcev BCL2-A1d in BCL2-S5b, ki smo ju podrobneje analizirali z NMR in komplementarnimi spektroskopskimi metodami. S CD-spektroskopijo smo potrdili, da obe zaporedji tvorita G-kvadrupleks paralelne topologije, z UV-spektroskopijo pa smo določili njuni talilni temperaturi, ki znašata kar 67,1 in 76,8 °C. Eksperiment DOSY je pokazal, da BCL2-S5b v raztopini tvori več različnih struktur, BCL2-A1d pa zgolj eno glavno strukturo, ki smo jo z NMRspektroskopijo določili kot trikvartetni G-kvadrupleks s 17-nukleotidno srednjo zanko. Titracije z ligandoma PhenDC3 in RHPS4, ki sta znana po selektivni vezavi na Gkvadruplekse, so pokazale, da PhenDC3 z obema G-kvadrupleksoma tvori kompleks, na drugi strani pa RHPS4 oba destabilizira. Na vzorcu BCL2-S5b smo pokazali tudi, da PhenDC3 interagira izključno z jedrom G-kvadrupleksa, kar bi lahko zagotavljalo dvojno specifičnost prek hkratnega ciljanja dolge zanke, v kateri se tvorijo Watson-Crickovi bazni pari. DNA is a highly dynamic carrier of genetic information which can form a variety of secondary structures beyond the canonical double helix. G-quadruplexes are among the best studied noncanonical structures, which emerge as potent regulators of gene expression. Most research on G-quadruplexes has been considering only motifs, which comply with the commonly accepted consensus sequence, however the focus has recently shifted towards alternative G-quadruplex formations. Long-looped G-quadruplexes are especially intriguing for their potential to indulge in highly specific interactions both with cellular proteins and small molecules – ligands. We have identified several potential long-looped G-quadruplexes in the guanine-rich BCL-2 promoter, which under experimental conditions used failed to fold in any relevant secondary structure due to the suboptimal motif selection. However, with specific modifications, we were able to determine crucial structural characteristics of long-looped G-quadruplexes. These include bulge-less G-tracts, which seem to be a predominant factor, and Watson-Crick base-pairing within the long loop, which gains significance with increasing loop length. During the process of optimizing the wild-type sequences we managed to create novel model sequences BCL2-A1d and BCL2-S5b, which we analysed is greater detail by NMR and complementary spectroscopic methods. Using CD spectroscopy, we confirmed that both samples form G-quadruplexes of parallel topology. Additionally, melting temperatures of 67.1 and 76.8 °C were determined with UV-melting experiments. DOSY spectra indicated that BCL2-A1d folds into a single major structure, while BCL2-S5b forms many structures. Using various methods of NMR spectroscopy, we determined that BCL2-A1d folds into a three-quartet G-quadruplex with a 17-nucleotide central loop. Titrating both samples with G-quadruplex specific ligand RHPS4 showed that it is not suitable for targeting these specific structures. On the other hand, ligand PhenDC3 at low concentrations formed a complex with both structures interacting only with the Gquadruplex core. This was especially evident with BCL2-S5b, where no changes of the Watson-Crick base-pairing of the central loop were observed, meaning that PhenDC3 could be used in dual-specific targeting approaches. DNA G-kvadrupleks dolge zanke BCL-2 NMR DNA G-quadruplex long loops BCL-2 NMR true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-08-31 09:55:47 2026-08-31 09:56:05 2026-09-15 13:12:46 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25934 291160835 Tanko_Lan_-_Karakterizacija_G-kvadrupleksov_z_dolgimi_zankami_v_promotorju_gena_BCL-2.pdf Tanko_Lan_-_Karakterizacija_G-kvadrupleksov_z_dolgimi_zankami_v_promotorju_gena_BCL-2.pdf 1 CC9D634E74A93A88D4AEBCD2B79E63C9 5c73812c796ad86cea6cf3fb2c2377a5e6263d1075bea19163842f0440de45cd 13f17557-a511-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245480 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0