20.500.12556/RUL-188475 Optimizacija šaržne reakcije transkripcije in vitro s kotranskripcijskim pokritjem 5’-konca mRNA Optimization of batch in vitro transcription reaction with co-transcriptional capping of mRNA Molekula informacijske RNA (mRNA, angl. messenger RNA) igra vlogo povezovalca med gensko informacijo, shranjeno v molekuli DNA, in proteini, za katere ta informacija zapisuje. Zaradi svojega prehodnega izražanja in nezmožnosti vključitve v gostiteljski genom je mRNA privlačna za uporabo v različne terapevtske namene, na primer kot cepivo, protirakava imunoterapija ali sredstvo celičnega reprogramiranja. Molekule mRNA sintetično proizvajamo s postopkom reakcije transkripcije in vitro (IVT, angl. in vitro transcription), ki je zaradi kompleksne narave in visokih stroškov primerna za optimizacijo s pristopom načrtovanja eksperimentov (DOE, angl. design of experiments). Pri diplomskem delu smo želeli reakcijo optimizirati tako, da bi z zmanjšanjem količine uporabljenega reagenta za kotranskripcijsko pokritje 5’‑konca mRNA CleanCap AG povečali stroškovno učinkovitost reakcije, obenem pa ohranili visok delež ustrezno pokritega produkta in omejili nastanek imunogenega stranskega produkta, dvoverižne RNA (dsRNA, angl. double‑stranded RNA). Pokritje 5’-konca mRNA s kapo je ključnega pomena za kakovost in varnost mRNA terapevtika, zato smo želeli doseči učinkovitost pokritosti s 5’-kapo vsaj 95 %. Za namen optimizacije smo izvedli 20 šaržnih reakcij IVT, v katerih smo proizvedli mRNA z zapisom za ojačani zeleni fluorescenčni protein (eGFP, angl. enhanced green fluorescent protein) in s pomočjo programa za načrtovanje eksperimentov MODDE preučili vpliv različnih parametrov na potek reakcije. Ugotovili smo, da lahko najboljše rezultate dosežemo z nizkim razmerjem med koncentracijo Mg2+ in koncentracijo NTP, ki nam omogoči manjšo porabo reagenta CleanCap AG ob visoki učinkovitosti pokritosti, obenem pa zmanjša vsebnost dsRNA in ne poslabša donosa reakcije. Določili smo tudi optimalno točko reakcije in jo uspešno validirali na konstruktu mRNA z zapisom za eGFP, delno pa tudi na konstruktih z zapisom za luciferazo in za protein bodice virusa SARS‑CoV‑2. Glede na količino pridobljene mRNA smo dosegli zmanjšanje porabe reagenta CleanCap AG za 25 % v primerjavi z originalnim protokolom proizvajalca reagenta. Messenger RNA (mRNA) acts as a link between the genetic information, stored in a DNA molecule, and the proteins for which this information codes. Due to its transient expression and inability to integrate into the host genome, mRNA is attractive for various therapeutic applications like vaccines, cancer immunotherapies, and cellular reprogramming. mRNA molecules are synthetically produced via in vitro transcription (IVT) reaction, which is, due to its complex nature and high cost, well‑suited for optimization with design of experiments (DOE) approach. In this bachelor's thesis, we wanted to optimize the reaction by reducing the amount of CleanCap Reagent AG used for co‑transcriptional capping of mRNA and subsequently increasing cost efficiency, while maintaining a high proportion of capped product and limiting the formation of immunogenic byproduct, double‑stranded RNA (dsRNA). As 5' capping is crucial for the quality and safety of mRNA therapeutics, our minimum requirement for capping efficiency was 95%. For the purpose of optimization, we performed 20 batch IVT reactions, producing mRNA coding for enhanced green fluorescent protein (eGFP), and investigated the effect of various reaction parameters using a DOE software, MODDE. We found that low ratio of Mg2+ and NTP concentations enables us to lower the consumption of CleanCap AG without sacrificing high capping efficiency. At the same time, the low ratio limits the formation of dsRNA and doesn’t seem to have a negative impact on reaction yield. Furthermore, we determined the optimal setpoint of the reaction and successfully validated it on mRNA construct coding for eGFP, and partially validated it on constructs coding for luciferase and SARS‑CoV‑2 spike protein. In regard to the amount of produced mRNA, we achieved a 25 % reduction in consumption of reagent CleanCap AG, compared to the original protocol of reagent manufacturer. mRNA IVT DOE optimizacija 5’‑kapa mRNA IVT DOE optimization 5’ capping true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-23 10:10:03 2026-09-23 10:10:10 2026-09-24 04:25:26 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-23 26045 Jordan_ferbezar_Uma_-_Optimizacija_sarzne_reakcije_transkripcije_in_vitro_s_kotranskripcijskim_p.pdf Jordan_ferbezar_Uma_-_Optimizacija_sarzne_reakcije_transkripcije_in_vitro_s_kotranskripcijskim_p.pdf 1 7F8E685FF308189E9CD34A0A50EF6BB1 f256bc6496664d2bd1d4c4795e84ab57bdec53210bc4985a6b0891381598af2f 210e4392-b726-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=248653 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0