Sinteza in vrednotenje fluorescenčnih sond na osnovi bisbenzimida za označevanje DNA

Potočnik, Benjamin (avtor), Pajk, Stane (mentor)

Fluorescenčna mikroskopija omogoča opazovanje analitov, označenih s fluorescenčnimi sondami – molekulami, ki po vzbujanju sevajo svetlobo. Na izbrani analit specifično vezano fluorescenčno sondo vzbudimo s svetlobo valovne dolžine, pri kateri absorbira v največjem obsegu, sevana svetloba pa ima zaradi energijskih izgub daljšo valovno dolžino. Razliko imenujemo Stokesov premik. Za učinkovito znotrajcelično opazovanje mora sonda prehajati celično membrano, se specifično vezati na izbrano celično komponento in se tam zadržati. Proces ne sme biti toksičen za celico, prav tako pa mora biti netoksično tudi samo vzbujanje, ki povzroča spremembe energijskega stanja. Ekstinkcijski koeficient ter kvantni izkoristek opredeljujeta, kako učinkovito sonda izkorišča prejeto svetlobo. Namen naloge je bil izboljšati lastnosti že uveljavljene fluorescenčne sonde Hoechst 33258, ki se specifično veže v mali jarek deoksiribonukleinske kisline (DNA). Njena glavna slabost je potreba po vzbujanju z ultravijolično (UV) svetlobo, kar negativno vpliva na obseg in uspešnost celične delitve. Optimizacijo molekule Hoechst 33258 smo skušali doseči z vezavo že obstoječih ali novo sintetiziranih fluoroforov prek dveh pristopov: s pripenjanjem neposredno na molekulo Hoechst 33258 ali preko različnih distančnikov. Delo smo pričeli s sintezo lastne spojine Hoechst 33258. Sintezna pot je opisana v literaturi, mi pa smo jo uspešno optimizirali za pripravo večjih količin produkta. Sledili so poskusi vezave fluoroforov na pridobljeni Hoechst 33258. Neposredno pripenjanje je bilo neuspešno, medtem ko smo z uporabo distančnika uspešno sintetizirali in izolirali spojino 18. Sinteza je ponovljiva, izolacija produkta pa nezahtevna. Spojino 18 smo uspešno uporabili za označevanje izolirane DNA, pri čemer smo zaznali povečanje fluorescence vezane oblike v primerjavi s prosto molekulo 18. Kasnejši poskusi nanosa spojine 18 na žive celice so pokazali, da molekula ne vstopa v znotrajcelični prostor. Potencial molekule 18 bi lahko izpopolnili z nadaljnjo optimizacijo distančnika ali fluorofora, ki smo ga pripeli na osnovno strukturo Hoechst 33258, in tako dosegli večji obseg vstopa sonde v celico in jedro ter povečali Stokesov premik.

• Jezik: Slovenski jezik
• Ključne besede: fluorescenca, Stokesov premik, fluorofor, Hoechst
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-170454
• Datum objave v RUL: 06.07.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis and evaluation of fluorescent probes based on bisbenzimide for DNA labelling

Izvleček

Fluorescence microscopy enables the observation of analytes labelled with fluorescent probes - molecules that emit light upon excitation. A fluorescent probe specifically bounds to the selected analyte is excited with light of a wavelength at which it absorbs most efficiently, while the emitted light has a longer wavelength due to energy loss. This difference is referred to as the Stokes shift. For effective intracellular observation, the probe must be capable of crossing the cell membrane, binding specifically to the selected cellular component, and remain localized at the target site. The entire process must be non-toxic to the cell, including the excitation step, which alters the energy state and could cause cellular damage. The extinction coefficient and quantum yield define how efficiently the probe utilizes the absorbed light. In this study, we aimed to improve the properties of the established fluorescent probe Hoechst 33258, which binds specifically to the minor groove of deoxyribonucleic acid (DNA). Its main drawback is the need for ultraviolet (UV) excitation, which negatively affects the extent and success of cell division. We attempt to optimize the Hoechst 33258 molecule by attaching either existing or newly synthesized fluorophores through two approaches: direct attachment to the Hoechst 33258 molecule or via various linkers. We began our work by synthesizing our own Hoechst 33258 compound. Although the synthetic route is described in the literature, we successfully optimized it for larger-scale laboratory synthesis. We then attempted to attach fluorophores to the synthesized Hoechst 33258. Direct conjugation was unsuccessful, whereas using a linker allowed us to successfully synthesize and isolate compound 18. The synthesis is reproducible, and the product isolation is straightforward. Compound 18 was successfully used to label isolated DNA, resulting in increased fluorescence of the bound form compared to the free molecule. A small Stokes shift was also observed. However, subsequent attempts to apply compound 18 to live cells showed that the molecule does enter cells. While the reason for this remains unclear, the inability to penetrate the membrane opens opportunities for further optimization of the linker or fluorophore.

• Ključne besede: fluorescence, Stokes shift, fluorophore, Hoechst