Stöðugar stakeindir fyrir lífeðlisfræðilegar rannsóknir - verkefni lokið
Fréttatilkynning verkefnisstjóra
Fréttatilkynning:
Stöðugar stakeindir fyrir lífeðlisfræðilegar rannsóknir
Verkefnisnúmer: 141062-051
Heiti verkefnis: Stöðugar stakeindir fyrir lífeðlisfræðilegar rannsóknir
Verkefnisstjóri: Snorri Þór Sigurðsson, prófessor, Háskóla Íslands
Kjarnsýrur gegna mörgum hlutverkum í lífverum. Upplýsingar um byggingu og hreyfingu kjarnsýra varpa ljósi á hvernig þær geta sinnt sínum fjölbreyttu hlutverkum. Tvær mæliaðferðir sem eru mjög gagnlegar til að safna slíkum upplýsingum eru kjarnsegulgreining (e. nuclear magnetic resonance, NMR) og rafeindasegulgreining (e. electron paramagnetic resonance, EPR). Meginmarkmið verkefnisins var að smíða og beita stöðugum stakeindum (spunamerkjum) við lífeðlisfræðilegar mælingar með NMR og EPR. Nýjar og betri aðferðir voru þróaðar til að festa slík spunamerki á ákveðna staði í RNA, bæði með og án samgildra tengja. Síðarnefnda aðferðin hefur þann kost að spunamerkingin er auðveld, spunamerkið og kjarnsýrunni er einfaldlega blandað saman í lausn. Spunamerki voru innleidd í nokkrar virkar RNA sameindir, til að mynda malachite green aptamerinn, tetracyclin aptamerinn og gúanín ríbórofann, sem tekur þátt í stjórnun á tjáningu gena. Þessar spunamerktu RNA sameindir er verið að rannsaka af samstarfsmönnum, bæði með NMR og EPR litrófsgreiningum. Nokkrar tvístakeindir voru einnig smíðaðar í því augnamiði að auka næmni NMR með mögnun á kjarnaskautun (e. dynamic nuclear polarization, DNP). Þar ber hæst nítroxíð tvístakeindir sem sýna mestu aukningu á næmni NMR mælinga til þessa, samanborið við önnur efni í flokki slíkra sameinda. Sú vinna leiddi til einkaleyfisumsóknar. Nýjar stakeindir sem valda DNP aukningu geta haft gífurleg áhrif á NMR mælingar á líffræðilegum sameindum, örögnum og yfirborðum efna, auk segulómunar á líffræðilegum vefjum og lífverum. Verkefnið var unnið í samstarfi við rannsóknahópa í Þýskalandi og Frakklandi.
Rannsóknasjóður Rannís styrkti verkefnið með rúmlega 88 m.kr. framlagi á þriggja ára tímabili. Raunvísindastofnun Háskólans og Rannsóknasjóður Háskóla Íslands styrktu einnig verkefnið.
Auk kynningar á niðurstöðum verkefnisins á ráðstefnum og við erlenda háskóla voru eftirtaldar greinar, einkaleyfisumsókn og ritgerðir birtar um niðurstöður verkefnis:
Greinar:
1. "TMIO-PyrImid hybrids are profluorescent, site-directed spin labels for nucleic acids," Chalmers BA, Saha S, Nguyen T, McMurtrie J, Sigurdsson ST, Bottle SE, Masters K-S, Org. Lett., 16, 5528-31 (2014).
2. Gutmann T, Liu J, Rothermel N, Xu Y, Jaumann E, Breitzke H, Sigurdsson ST, Buntkowsky G, "Natural abundance N15 NMR by dynamic nuclear polarization: Fast analysis of binding sites of a novel amine-carboxyl-linked immobilized dirhodium catalyst," Chem. Eur J., 21, 3798-805 (2015).
3. Saha S, Jagtap AP, Sigurdsson ST, "Site-directed spin labeling of 2′-amino groups in RNA with isoindoline nitroxides that are resistant to reduction," Chem. Comm. 51, 13142-5 (2015).
4. Jagtap AP, Geiger M-A, Stöppler D, Orwick-Rydmark M, Oschkinat H, Sigurdsson ST, "bcTol: A highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules," Chem. Comm. 52, 7020-3 (2016).
5. Kamble NR, Gränz M, Prisner TF, Sigurdsson ST, "Noncovalent and site-directed spin labeling of duplex RNA,"Chem. Comm, 52, 14442-5 (2016).
6. Stöppler D, Song C, van Rossum B-J, Geiger M-A, Lang C, Mroginski M-A, Jagtap AP, Sigurdsson ST, Matysik J, Hughes J, Oschkinat H, "Dynamic nuclear polarization provides new insights into chromophore structure in phytochrome photoreceptors," Angew. Chem. Int. Ed.. 55, 16017-16020 (2016).
7. Schnorr K, Gophane DB, Helmling C, Cetiner E, Pasemann K, Fürtig B, Wacker A, Qureshi NS, Gränz M, Barthelmes D, Jonker HRA, Stirnal E, Sigurdsson ST, Schwalbe H, "Impact of spin label rigidity on extent and accuracy of distance information from PRE data," J. Biomol. NMR, 68, 53-63 (2017).
8. Thankamony A, Knoche S, Bothe S, Drochner A, Jagtap AP, Sigurdsson ST, Vogel H, Etzold B, Gutmann T, Buntkowsky G, "Characterization of V-Mo-W mixed oxide catalyst surface species by 51V solid-state dynamic nuclear polarization NMR," J. Phys. Chem. C., 121, 20857–64 (2017).
9. Gophane DB, Endeward B, Prisner TF, Sigurdsson ST, "A semi-rigid isoindoline-derived nitroxide spin label for RNA," Org. Biomol. Chem. 16, 816-24 (2018).
10. Scott FJ, Saliba EP, Albert BJ, Alaniva N, Sesti EL, Gao C, Golota NC, Choi EJ, Jagtap AP, Whitman JJ, Eckardt M, Harneit W, Corzilius B, Sigurdsson ST, Barnes AB, "Frequency-agile gyrotron for electron decoupling and pulsed dynamic nuclear polarization," J. Magn. Reson. 289, 45-54 (2018).
11. Kamble NR, Sigurdsson ST, "Purine-derived nitroxides for noncovalent spin-labeling of abasic sites in duplex nucleic acids," Eur J. Chem. 24, 4157-4164 (2018).
12. Geiger M-A, Jagtap AP, Kaushik M, Sun H, Sigurdsson ST, Corzilius B, Oschkinat H, "Efficiency of water-soluble nitroxide biradicals for dynamic nuclear polarization in rotating solids at 9.4 T: bcTol-M and cyolyl-TOTAPOL as new polarizing agents," submitted (2018).
13. Mentink-Vigier F, Marin-Montesinos I, Jagtap AP, Halbritter T, van Tol J, Hediger S, Lee D, Sigurdsson ST, De Paëpe G, " AsymPol: a family of simple asymmetric bis-nitroxides for efficient dynamic nuclear polarization," submitted (2018).
14. Gränz M, Erlenbach N, Spindler P, Gophane DB, Stelzl LS, Sigurdsson ST, Prisner TF, "Dynamics of nucleic acids at room temperature revealed by pulsed EPR," submitted (2018).
Einkaleyfi:
1. Mentink-Vigier F, Jagtap AP, Sigurdsson ST, De Paëpe G, "Dinitroxide radical compounds as polarizing agents," EP application no. 17179869.7-1568, filed 05.07.2017.
Ritgerðir:
1. "Syntheses of biradicals for surface studies by DNP ssNMR," Snædís Björgvinsdóttir, MS thesis, University of Iceland (2016).
2. "Water-soluble nitroxides for dynamic nuclear polarization," Anil P Jagtap, PhD thesis, University of Iceland (2016).
3. "Advancement of covalent and noncovalent nitroxide spin-labeling of RNA," Subham Saha, PhD thesis, University of Iceland (2016).
4. "Purine-derived nitroxides for noncovalent spin-labeling of abasic sites in duplex nucleic acids," Nilesh R Kamble, PhD thesis, University of Iceland (2017).
Press-release: Stable
radicals for biophysical studies
Grant number: 141062-051
Project name: Stable radicals for biophysical studies
Principal investigator: Snorri Þór Sigurðsson, professor, University of Iceland
Nucleic acids have many important roles in cellular function. In order to understand how how nucleic acids are able to carry out their functions, information about their structure and dynamics is required. Two techniques that are valuable for obtaining such information are nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies. The purpose of this project was the preparation and application of stable radicals (spin labels) for biophysical studies with NMR and EPR. New and improved methods were developed for incorporation of radicals at specific sites in RNA, both by formation of covalent bonds between the RNA and the spin labels, as well as by non-covalent bonding. The latter method allows an easy sample preparation, as the spin label and the nucleic acid are simply mixed together in solution. Spin labels were incorporated into different functional RNAs, namely the malachite green aptamer, the tetracycline aptamer and the guanosine riboswitch, which is involved in gene regulation. These spin-labeled RNAs are currently being investigated by collaborators using both NMR and EPR spectroscopies. A number of stable biradicals were also prepared with the aim of increasing the sensitivity of NMR through dynamic nuclear polarization (DNP). Specifically, nitroxide biradicals were prepared that show the greatest signal enhancement reported thus far for this class of compounds. One patent application has been filed as a result of this work. New and improved radicals for DNP could have an enormous impact on NMR studies of biologically relevant molecules, materials, such as nanoparticles and surface chemistry, as well as magnetic resonance imaging (MRI) of biological tissues and organisms. This research project was carried out in collaboration with research groups in Germany and France.
The Icelandic Research Fund provided a ca. ISK 80 million in financial support for this project over a period of three years.
In addition to presentation of the results of this project in international conferences and at different universities, it also resulted in the following articles, a patent application and theses:
Articles:
1. "TMIO-PyrImid hybrids are profluorescent, site-directed spin labels for nucleic acids," Chalmers BA, Saha S, Nguyen T, McMurtrie J, Sigurdsson ST, Bottle SE, Masters K-S, Org. Lett., 16, 5528-31 (2014).
2. Gutmann T, Liu J, Rothermel N, Xu Y, Jaumann E, Breitzke H, Sigurdsson ST, Buntkowsky G, "Natural abundance N15 NMR by dynamic nuclear polarization: Fast analysis of binding sites of a novel amine-carboxyl-linked immobilized dirhodium catalyst," Chem. Eur J., 21, 3798-805 (2015).
3. Saha S, Jagtap AP, Sigurdsson ST, "Site-directed spin labeling of 2′-amino groups in RNA with isoindoline nitroxides that are resistant to reduction," Chem. Comm. 51, 13142-5 (2015).
4. Jagtap AP, Geiger M-A, Stöppler D, Orwick-Rydmark M, Oschkinat H, Sigurdsson ST, "bcTol: A highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules," Chem. Comm. 52, 7020-3 (2016).
5. Kamble NR, Gränz M, Prisner TF, Sigurdsson ST, "Noncovalent and site-directed spin labeling of duplex RNA,"Chem. Comm, 52, 14442-5 (2016).
6. Stöppler D, Song C, van Rossum B-J, Geiger M-A, Lang C, Mroginski M-A, Jagtap AP, Sigurdsson ST, Matysik J, Hughes J, Oschkinat H, "Dynamic nuclear polarization provides new insights into chromophore structure in phytochrome photoreceptors," Angew. Chem. Int. Ed.. 55, 16017-16020 (2016).
7. Schnorr K, Gophane DB, Helmling C, Cetiner E, Pasemann K, Fürtig B, Wacker A, Qureshi NS, Gränz M, Barthelmes D, Jonker HRA, Stirnal E, Sigurdsson ST, Schwalbe H, "Impact of spin label rigidity on extent and accuracy of distance information from PRE data," J. Biomol. NMR, 68, 53-63 (2017).
8. Thankamony A, Knoche S, Bothe S, Drochner A, Jagtap AP, Sigurdsson ST, Vogel H, Etzold B, Gutmann T, Buntkowsky G, "Characterization of V-Mo-W mixed oxide catalyst surface species by 51V solid-state dynamic nuclear polarization NMR," J. Phys. Chem. C., 121, 20857–64 (2017).
9. Gophane DB, Endeward B, Prisner TF, Sigurdsson ST, "A semi-rigid isoindoline-derived nitroxide spin label for RNA," Org. Biomol. Chem. 16, 816-24 (2018).
10. Scott FJ, Saliba EP, Albert BJ, Alaniva N, Sesti EL, Gao C, Golota NC, Choi EJ, Jagtap AP, Whitman JJ, Eckardt M, Harneit W, Corzilius B, Sigurdsson ST, Barnes AB, "Frequency-agile gyrotron for electron decoupling and pulsed dynamic nuclear polarization," J. Magn. Reson. 289, 45-54 (2018).
11. Kamble NR, Sigurdsson ST, "Purine-derived nitroxides for noncovalent spin-labeling of abasic sites in duplex nucleic acids," Eur J. Chem. 24, 4157-4164 (2018).
12. Geiger M-A, Jagtap AP, Kaushik M, Sun H, Sigurdsson ST, Corzilius B, Oschkinat H, "Efficiency of water-soluble nitroxide biradicals for dynamic nuclear polarization in rotating solids at 9.4 T: bcTol-M and cyolyl-TOTAPOL as new polarizing agents," submitted (2018).
13. Mentink-Vigier F, Marin-Montesinos I, Jagtap AP, Halbritter T, van Tol J, Hediger S, Lee D, Sigurdsson ST, De Paëpe G, " AsymPol: a family of simple asymmetric bis-nitroxides for efficient dynamic nuclear polarization," submitted (2018).
14. Gränz M, Erlenbach N, Spindler P, Gophane DB, Stelzl LS, Sigurdsson ST, Prisner TF, "Dynamics of nucleic acids at room temperature revealed by pulsed EPR," submitted (2018).
Patents:
1. Mentink-Vigier F, Jagtap AP, Sigurdsson ST, De Paëpe G, "Dinitroxide radical compounds as polarizing agents," EP application no. 17179869.7-1568, filed 05.07.2017.
Theses:
1. "Syntheses of biradicals for surface studies by DNP ssNMR," Snædís Björgvinsdóttir, MS thesis, University of Iceland (2016).
2. "Water-soluble nitroxides for dynamic nuclear polarization," Anil P Jagtap, PhD thesis, University of Iceland (2016).
3. "Advancement of covalent and noncovalent nitroxide spin-labeling of RNA," Subham Saha, PhD thesis, University of Iceland (2016).
4. "Purine-derived nitroxides for noncovalent spin-labeling of abasic sites in duplex nucleic acids," Nilesh R Kamble, PhD thesis, University of Iceland (2017).eland (2017).
