Stíf merki og greiningar á byggingu og hreyfingu kjarnsýra með litrófsaðferðum - verkefni lokið
Fréttatilkynning verkefnisstjóra
Meginmarkmið verkefnisins var að smíða stöðugar stakeindir (spunamerki) og flúrljómunarmerki og beita þeim við lífeðlisfræðilegar mælingar á kjarnsýrum með EPR- og flúrljómunar-litrófsgreiningum. Nýjar og betri aðferðir voru þróaðar til að festa spunamerki á ákveðna staði í DNA og RNA.
Kjarnsýrur gegna mörgum hlutverkum í lífverum. Upplýsingar um byggingu og hreyfingu kjarnsýra varpa ljósi á hvernig þær geta sinnt sínum fjölbreyttu hlutverkum. Tvær litrófsgreiningaraðferðir sem eru mjög gagnlegar til að safna slíkum upplýsingum eru flúrljómunarmælingar og rafeindasegulgreining (e. electron paramagnetic resonance, EPR).
Spunamerki voru innleidd seríu RNA tvíliða til rannsókna á innri hreyfnigu þeirra. EPR gögnin voru notuð til að sýna fram á að aðeins eitt kraftsvið (e. force field) fyrir sameindahreyfihermanir (e. molecular dynamics simulation, MD), af nokkrum þekktum kraftsviðum, gat framkallað á ný gögnin úr EPR tilraununum. Þessar niðurstöður munu hafa áhrif á slíka útreikninga á RNA í framtíðinni. Tetracyclin RNA aptamerinn var einnig spunamerktur og EPR greining sýndi fram á hlutverk Mg2+ jóna við myndun þrívíddarbyggingarinnar; við háan Mg2+ styrk myndaðist virka byggingin án tetracycline tengilsins. Flúrljómandi merki voru einnig innleidd í neomycin aptamerinn. Nokkrar tegundir flúrljómunarmælinga voru notaðar til að sýna fram á að neomycin aptamerinn binst tengli sínum, sýklalyfinu neomycin, í tveimur skrefum. Þessar niðurstöður varpa ljósi á hvernig RNA binst litlum sameindum en margar líffræðilegar RNA sameindir bindast slíkum tenglum og geta með því stjórnað genatjáningu. Verkefnið var unnið í samstarfi við rannsóknahópa í Þýskalandi.
Auk kynningar á niðurstöðum verkefnisins á ráðstefnum og við erlenda háskóla voru eftirtaldar greinar og ein doktorsritgerð birt um niðurstöður verkefnis:
Greinar í ritrýndum tímaritum:
1.
"Influence
of Mg2+ on the conformational flexibility of a tetracycline aptamer,"
Hetzke T, Vogel M, Gophane DB, Weigand JE, Suess B, Sigurdsson ST, Prisner TF, RNA 2019, 25,
158-167.
2. "Benzoyl‐protected hydroxylamines for improved chemical synthesis of oligonucleotides containing nitroxide spin labels," Juliusson HY, Segler A-LJ, Sigurdsson ST, Eur. J. Org Chem 2019, 23, 3799-805.
3. "Ligand-binding dynamics of the neomycin aptamer with Çmf, an RNA fluorescence label," Gustmann H, Segler A-LJ, Gophane DB, Reuss AJ, Grünewald C, Braun M, Sigurdsson ST, Wachtveitl J, Nucleic Acids Res. 2019, 47, 15-28.
4. "Reduction resistant and rigid spin labels for DNA and RNA," Juliusson HY, Sigurdsson ST, J. Org. Chem. 2020, 85, 4036-46.
5. "Nitroxide-derived N-oxide phenazines for noncovalent spin-labeling of DNA," Juliusson HY, Sigurdsson ST, ChemBioChem 2020, 21, 2635-42.
6. "Noncovalent spin‐labeling of DNA and RNA triplexes," Richert C, Kamble N, Wolfrum M, Halbritter T, Sigurdsson ST, Chem. Biodiversity 2020, 17, e1900676.
7. "Internal dynamics of RNA duplexes," Gauger M, Segler A-LJ, Erlenbach N, Stelzl L, Hummer G, Sigurdsson ST, Prisner T, handrit í vinnslu, 2021.
Doktorsritgerð:
1. "Advancements in site-directed incorporation of rigid spin labels into nucleic acids," Haraldur Yngvi Júlíusson, doktorsritgerð, Háskóli Íslands (2020).
English:
Nucleic acids have many important roles in cellular function. In order to understand how nucleic acids are able to carry out their functions, information about their structure and dynamics is required. Two valuable techniques for obtaining such information are fluorescence spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. The purpose of this project was the preparation and application of persistent radicals (spin labels) and fluorescent labels for biophysical studies of nucleic acids with EPR and fluorescence spectroscopies, respectively. New and improved methods were implemented for incorporation of radicals at specific sites into both DNA and RNA. In particular, a protecting group strategy for nitroxides was developed, which enables chemical incorporation of nitroxide spin labels into nucleic acids without any reduction of the spin label during the nucleic acid synthesis. Spin labels were incorporated into a series of duplex RNAs to study their internal dynamics. This EPR data was used to show that only one of the available forcefields for molecular dynamic simulations of RNA can reproduce the experimental data. This will impact future in-silico studies of RNAs in folding and function. Furthermore, the RNA tetracycline aptamer was spin-labeled and subsequent EPR-studies shed light on its Mg2+-dependent folding; at high Mg2+-concentration, the ligand is not required for folding into its active structure. Fluorescent labels were also incorporated into the neomycin aptamer. Using several fluorescence spectroscopy techniques, we showed that the neomycin aptamer binds to its ligand, the antibiotic neomycin, in a two-step mechanism. This knowledge improves our understanding of how RNA binds to small molecules, but many biologically RNAs bind to such ligands and in doing so regulate gene expression. This research project was carried out in collaboration with research groups in Germany.
In addition to presentation of the results of this project in international conferences and at different universities, it also resulted in the following articles and one PhD thesis:
Articles in peer-reviewed journals:
1.
"Influence
of Mg2+ on the conformational flexibility of a tetracycline aptamer,"
Hetzke T, Vogel M, Gophane DB, Weigand JE, Suess B, Sigurdsson ST, Prisner TF, RNA 2019, 25,
158-167.
2. "Benzoyl‐protected hydroxylamines for improved chemical synthesis of oligonucleotides containing nitroxide spin labels," Juliusson HY, Segler A-LJ, Sigurdsson ST, Eur. J. Org Chem 2019, 23, 3799-805.
3. "Ligand-binding dynamics of the neomycin aptamer with Çmf, an RNA fluorescence label," Gustmann H, Segler A-LJ, Gophane DB, Reuss AJ, Grünewald C, Braun M, Sigurdsson ST, Wachtveitl J, Nucleic Acids Res. 2019, 47, 15-28.
4. "Reduction resistant and rigid spin labels for DNA and RNA," Juliusson HY, Sigurdsson ST, J. Org. Chem. 2020, 85, 4036-46.
5. "Nitroxide-derived N-oxide phenazines for noncovalent spin-labeling of DNA," Juliusson HY, Sigurdsson ST, ChemBioChem 2020, 21, 2635-42.
6. "Noncovalent spin‐labeling of DNA and RNA triplexes," Richert C, Kamble N, Wolfrum M, Halbritter T, Sigurdsson ST, Chem. Biodiversity 2020, 17, e1900676.
7. "Internal dynamics of RNA duplexes," Gauger M, Segler A-LJ, Erlenbach N, Stelzl L, Hummer G, Sigurdsson ST, Prisner T, manuscript in preparation, 2021.
Thesis:
1. "Advancements in site-directed incorporation of rigid spin labels into nucleic acids," Haraldur Yngvi Júlíusson, PhD thesis, University of Iceland (2020).
Heiti verkefnis:
Stíf merki og greiningar á byggingu og hreyfingu kjarnsýra
með litrófsaðferðum/ Rigid labels and spectroscopic analysis
of nucleic acid structure and dynamics
Verkefnisstjóri: Snorri Þór Sigurðsson, Raunvísindastofnun
Tegund styrks: Verkefnisstyrkur
Styrktímabil: 2017-2019
Fjárhæð styrks: 56,03 millj. kr. alls
Tilvísunarnúmer Rannís:
173727-053
