20.500.12556/RUL-186723 Prepoznavanje DNA s ponovitvami TCGCTCCC z nanotelesom iMbody Recognition of DNA Containing TCGCTCCC Repeats by the iMbody nanobody I-motivi so štiriverižne nekanonične strukture DNA, ki nastanejo iz s citozini bogatih zaporedij in jih stabilizirajo hemiprotonirani bazni pari citozin-citozin+. Imajo biološke funkcije pri uravnavanju prepisovanja genov, vključno s številnimi onkogeni, zaradi česar je zanesljiva detekcija i-motivov ključna, a doslej temelji pretežno na protitelesu iMab in njegovi novejši alternativi nanotelesu iMbody. V nedavnih študijah pa se je pojavil dvom o njuni strukturni specifičnosti. V diplomskem delu smo proučili interakcijo nanotelesa iMbody z modelnim DNA oligonukleotidom s ponovitvami TCGCTCCC, imenovanim VK2c, v pogojih, kjer je ta zvit v i-motiv in v pogojih, kjer je razvit. Strukturo DNA smo spremljali s cirkularno dikroično (CD) spektroskopijo, vezavo pa termodinamsko ovrednotili z izotermno titracijsko kalorimetrijo (ITC), oboje v acetatnem pufru pri pH 5 in v fosfatnem pufru pri pH 7. S pH titracijo v obeh smereh smo potrdili, da VK2c pri pH 5 zavzame značilno strukturo i-motiva, pri pH 7 pa je pretežno razvit, tako da izbrani pH vrednosti dobro predstavljata obe stanji. ITC je pokazala eksotermno, entalpijsko gnano vezavo pri obeh vrednostih pH, s konstanto disociacije 7,7 nM pri pH 5 in 471 nM pri pH 7 ter približno trikrat večjo navidezno stehiometrijo pri pH 7. CD spektri kompleksov nanotelesa in oligonukleotida niso bili aditivni, odstopanje pri pH 5 pa je bilo bistveno večje kot pri pH 7; spekter se ob prebitku nanotelesa ni pomikal proti razviti obliki, temveč nasprotno, k daljšim valovnim dolžinam. Nanotelo iMbody torej i-motiv prepozna prednostno, ne pa izključno, zaradi česar samo po sebi ni zanesljivo orodje za strukturno detekcijo i-motivov. I-motifs are four-stranded non-canonical DNA structures formed by cytosine-rich sequences and stabilised by hemiprotonated cytosine-cytosine+ base pairs. They have biological functions relating to the transcription of genes, including many oncogenes, meaning the reliable detection of i-motifs is essential, yet it is currently largely based on the iMab antibody and its newer alternative, the iMbody nanobody. Recent studies, however, have raised doubts about their structural specificity. In this thesis we investigated the interaction between the iMbody nanobody and the model oligonucleotide containing TCGCTCCC repeats, named VK2c, under conditions in which the oligonucleotide is folded into an i-motif and conditions under which it is unfolded. The structure of the DNA was monitored by circular dichroism (CD) spectroscopy, and the binding was characterised thermodynamically by isothermal titration calorimetry (ITC), both at pH 5 in acetate buffer and at pH 7 in phosphate buffer. A pH titration carried out in both directions confirmed that the oligonucleotide adopts the characteristic i-motif structure at pH 5 and is predominantly unfolded at pH 7, so that the two selected pH values represent the two states well. ITC revealed exothermic, enthalpy driven binding at both pH values, with a dissociation constant of 7.7 nM at pH 5 and 471 nM at pH 7, with an apparent stoichiometry about three times higher at pH 7. The CD spectra of the nanobody and oligonucleotide complexes were not additive, with a considerably larger deviation at pH 5 than at pH 7; in the presence of excess nanobody the spectrum did not shift towards the unfolded form, but in the opposite direction, towards longer wavelengths. The iMbody nanobody therefore recognizes the i-motif preferentially but not exclusively, which makes iMbody alone an unreliable tool for the structural detection of i-motifs. i-motiv nekanonične strukture DNA nanotelo iMbody cirkularni dikroizem izotermna titracijska kalorimetrija i-motif non-canonical DNA structures iMbody nanobody circular dichroism isothermal titration calorimetry true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-04 10:00:04 2026-09-04 10:00:20 2026-09-05 04:34:15 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 26145 Lenart_David_-_Prepoznavanje_DNA_s_ponovitvami_TCGCTCCC_z_nanotelesom_iMbody.pdf Lenart_David_-_Prepoznavanje_DNA_s_ponovitvami_TCGCTCCC_z_nanotelesom_iMbody.pdf 1 743B7449646AEDCAD7125C1D0CE56F58 3c12f27e754d5528fe3d5c3112a1740ce0b81b4a29f5241bcef1036991153ba9 ac064c73-a836-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246062 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0