TY - JOUR AU - Albert, Pál AU - Varga, Bence AU - Ferenc, Györgyi AU - Kiss, Antal TI - Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites JF - NUCLEIC ACIDS RESEARCH J2 - NUCLEIC ACIDS RES VL - 52 PY - 2024 SP - 1896 EP - 1908 PG - 13 SN - 0305-1048 DO - 10.1093/nar/gkad1217 UR - https://m2.mtmt.hu/api/publication/34547384 ID - 34547384 N1 - Funding Agency and Grant Number: Ministry of Finance of Hungary [GINOP-2.3.2-15-2016-00001]; Eotvoes Lorand Research Network (ELKH) [SA-110/2021] Funding text: Ministry of Finance of Hungary [GINOP-2.3.2-15-2016-00001]; Eotvoes Lorand Research Network (ELKH) [SA-110/2021]. Funding for open access charge: Budget of our institute. AB - We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5 '-CG/5 '-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (Delta A323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3 ' flanking position making it a CCA and CCC specific DNA methyltransferase. Graphical Abstract LA - English DB - MTMT ER - TY - JOUR AU - Nagy, Bettina AU - Öktem, Ayşegül AU - Ferenc, Györgyi AU - Ungor, Ditta Anita AU - Kalac, Aladina AU - Kelemen-Valkony, Ildikó AU - Ayaydin-Fodor, Elfrieda AU - Nagy, István AU - Dudits, Dénes AU - Ayaydin, Ferhan TI - CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES J2 - INT J MOL SCI VL - 24 PY - 2023 IS - 22 PG - 12 SN - 1661-6596 DO - 10.3390/ijms242216137 UR - https://m2.mtmt.hu/api/publication/34288968 ID - 34288968 N1 - Funding Agency and Grant Number: National Research, Development and Innovation Office of the Hungarian Government Funding text: No Statement Available AB - Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., “polyplexes” that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches. LA - English DB - MTMT ER - TY - JOUR AU - Dudits, Dénes AU - Cseri, András AU - Török, Katalin AU - Vankova, Radomira AU - Dobrev, Petre I. AU - Sass, László AU - Steinbach, Gábor AU - Kelemen-Valkony, Ildikó AU - Zombori, Zoltán AU - Ferenc, Györgyi AU - Ayaydin, Ferhan TI - Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants JF - GENES J2 - GENES-BASEL VL - 14 PY - 2023 IS - 10 PG - 17 SN - 2073-4425 DO - 10.3390/genes14101929 UR - https://m2.mtmt.hu/api/publication/34201709 ID - 34201709 N1 - Funding Agency and Grant Number: Hungarian government [GINOP-2.2.1-15-2017-00081, 2020-1.1.2-PIACI-KFI-2020-00117]; European Union [739593] Funding text: This work was funded by grant no. GINOP-2.2.1-15-2017-00081 and 2020-1.1.2-PIACI-KFI-2020-00117 from the Hungarian government. F.A. received support from the European Union's Horizon 2020 research and innovation program with grant number 739593. AB - Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63–19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division–elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin–cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots. LA - English DB - MTMT ER - TY - JOUR AU - Zombori, Zoltán AU - Török, Szabolcs AU - Nagy, Bettina AU - László, Nikolett AU - Sass, László AU - Jancsó, Mihály AU - Szabó, Gábor AU - Rádi, Feríz AU - Ferenc, Györgyi AU - Gyuricza, Csaba AU - Dudits, Dénes TI - Expression of triploid heterosis in the biomass productivity of energy willow plants under salinity stress JF - BIOMASS & BIOENERGY J2 - BIOMASS BIOENERGY VL - 174 PY - 2023 PG - 10 SN - 0961-9534 DO - 10.1016/j.biombioe.2023.106852 UR - https://m2.mtmt.hu/api/publication/33999425 ID - 33999425 N1 - Funding Agency and Grant Number: Hungarian government [2020-1.1.2-PIACI-KFI-2020-00117]; project 'Preparation for the transition to circular economy in the case of agricultural and green waste' of Environment and Energy Efficiency Operational Programme grant scheme of Ministry for Technology and Industry, Hungary [KEHOP-3.2.1-15-2021-00037] Funding text: This work was funded by grant 2020-1.1.2-PIACI-KFI-2020-00117 from the Hungarian government.The research was supported by the project 'Preparation for the transition to circular economy in the case of agricultural and green waste' of Environment and Energy Efficiency Operational Programme grant scheme of Ministry for Technology and Industry, Hungary under grant no.: KEHOP-3.2.1-15-2021-00037. LA - English DB - MTMT ER - TY - JOUR AU - Szeltner, Zoltán AU - Ferenc, Györgyi AU - Juhász, Tünde AU - Kupihár, Zoltán AU - Váradi, Zoltán AU - Szüts, Dávid AU - Kovács, Lajos TI - Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions JF - BIOCHIMIE J2 - BIOCHIMIE VL - 214 PY - 2023 IS - Part A SP - 33 EP - 44 PG - 12 SN - 0300-9084 DO - 10.1016/j.biochi.2023.01.009 UR - https://m2.mtmt.hu/api/publication/33697937 ID - 33697937 N1 - Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudósok Körútja 2, Budapest, H-1117, Hungary Nucleic Acid Synthesis Laboratory, Biological Research Centre, Eötvös Loránd Research Network, Temesvári Krt. 62, Szeged, H-6726, Hungary Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Magyar Tudósok Körútja 2, Budapest, H-1117, Hungary Department of Medicinal Chemistry, University of Szeged, Dom Tér 8, Szeged, H-6720, Hungary Export Date: 13 March 2023 CODEN: BICMB Correspondence Address: Szüts, D.; Institute of Enzymology, Magyar Tudósok Körútja 2, Hungary; email: szuts.david@ttk.hu Correspondence Address: Kovács, L.; Department of Medicinal Chemistry, Dom Tér 8, Hungary; email: kovacs.lajos@med.u-szeged-hu Funding details: Eötvös Loránd Tudományegyetem, ELTE Funding details: National Research, Development and Innovation Office, 2018–1.2.1-NKP-2018-00005, K128801 Funding text 1: We thank Mihály Kovács and Gábor Harami (ELTE) for supplying us with BLM helicase. This work was supported by the National Research Development and Innovation Office of Hungary (grants no. K128801 and 2018–1.2.1-NKP-2018-00005 ). Funding text 2: We thank Mihály Kovács and Gábor Harami (ELTE) for supplying us with BLM helicase. This work was supported by the National Research Development and Innovation Office of Hungary (grants no. K128801 and 2018–1.2.1-NKP-2018-00005). AB - Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states. © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM) LA - English DB - MTMT ER - TY - JOUR AU - Kupihár, Zoltán AU - Ferenc, Györgyi AU - Petrovicz, Vencel László AU - Fáy, Viktória R. AU - Kovács, Lajos AU - Martinek, Tamás AU - Hegedüs, Zsófia TI - Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates JF - PHARMACEUTICS J2 - PHARMACEUTICS VL - 15 PY - 2023 IS - 1 PG - 16 SN - 1999-4923 DO - 10.3390/pharmaceutics15010248 UR - https://m2.mtmt.hu/api/publication/33597958 ID - 33597958 N1 - Funding Agency and Grant Number: National Research, Development and Innovation Office of Hungary [NKFIH PD 135324, K 128801, K 134754]; Ministry of Innovation and Technology of Hungary through the National Research, Development and Innovation Fund [TKP2021-EGA-32] Funding text: This work received funding from the National Research, Development and Innovation Office of Hungary-NKFIH PD 135324 (Z.H.), K 128801 (L.K.) and K 134754 (T.A.M.). Support by the Ministry of Innovation and Technology of Hungary through the National Research, Development and Innovation Fund (TKP2021-EGA-32) is acknowledged. AB - Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5′ or 3′ end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation. LA - English DB - MTMT ER - TY - JOUR AU - Bashir, Faiza AU - Kovács, Sándor AU - Ábrahám, Ágnes AU - Nagy, Krisztina AU - Ayaydin, Ferhan AU - Kelemen-Valkony, Ildikó AU - Ferenc, Györgyi AU - Galajda, Péter AU - Tóth, Szilvia Zita AU - Sass, László AU - Kós, Péter AU - Vass, Imre AU - Szabó, Milán TI - Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform JF - LAB ON A CHIP J2 - LAB CHIP VL - 22 PY - 2022 IS - 16 SP - 2986 EP - 2999 PG - 14 SN - 1473-0197 DO - 10.1039/D2LC00130F UR - https://m2.mtmt.hu/api/publication/32832922 ID - 32832922 N1 - Funding Agency and Grant Number: Hungarian Academy of Sciences, MTA Premium Postdoctoral Research Program [GINOP-2.3.2-15-2016-00026, PREMIUM-2017-38]; National Research, Development and Innovation Office [NKFIH FK 128977, NKFIH K 116016, NKFIH K 116526, NKFIH PD 124889]; Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences [BO/00463/18/8] Funding text: This work was supported by the grants GINOP-2.3.2-15-2016-00026, Hungarian Academy of Sciences, MTA Premium Postdoctoral Research Program PREMIUM-2017-38 (awarded to M. S.), National Research, Development and Innovation Office grant number NKFIH FK 128977 (awarded to M. S.), NKFIH K 116016, NKFIH K 116526 (awarded to P. G.), and NKFIH PD 124889 (awarded to K. N.) K. N. was supported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00463/18/8). The authors thank Laszlo Der for valuable discussions and his help with the model calculations. AB - Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress. LA - English DB - MTMT ER - TY - JOUR AU - Dudits, Dénes AU - Cseri, András AU - Török, Katalin AU - Sass, László AU - Zombori, Zoltán AU - Ferenc, Györgyi AU - Poór, Péter AU - Borbély, Péter Gábor AU - Czékus, Zalán AU - Vankova, Radomira AU - Dobrev, Petre AU - Szántó, Judit AU - Bagi, Zoltán AU - Kovács, Kornél Lajos TI - Triploid Hybrid Vigor in Above-Ground Growth and Methane Fermentation Efficiency of Energy Willow JF - FRONTIERS IN PLANT SCIENCE J2 - FRONT PLANT SCI VL - 13 PY - 2022 PG - 14 SN - 1664-462X DO - 10.3389/fpls.2022.770284 UR - https://m2.mtmt.hu/api/publication/32722153 ID - 32722153 N1 - Institute of Plant Biology, Biological Research Centre, Eötvös Loránd Research Network (ELKH), Szeged, Hungary Department of Plant Biology, University of Szeged, Szeged, Hungary Department of Biological Resources, Centre for Agricultural Research, Agricultural Institute, Martonvásár, Hungary Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic UBM Feed Zrt. Pilisvörösvár, Hungary Department of Biotechnology, University of Szeged, Szeged, Hungary Export Date: 6 April 2022 Correspondence Address: Ferenc, G.; Institute of Plant Biology, Hungary; email: ferenc.gyorgyi@brc.hu Funding details: Magyarország Kormánya Funding text 1: This study was funded by grant nos. GINOP-2.2.1-15-2017-00081 and 2020-1.1.2-PIACI-KFI-2020-00117 from the Hungarian Government. The authors are very grateful to Feríz Rádi (Kiskun Kft. Kiskunhalas, Hungary) for providing the experimental field. LA - English DB - MTMT ER - TY - JOUR AU - Veszelka, Szilvia AU - Mészáros, Mária AU - Porkoláb, Gergő AU - Szecskó, Anikó AU - Kondor, Nóra AU - Ferenc, Györgyi AU - Polgár, Tamás Ferenc AU - Katona, Gábor AU - Kóta, Zoltán AU - Kelemen, Lóránd AU - Páli, Tibor AU - Vigh, Judit Piroska AU - Walter, Fruzsina AU - Bolognin, Silvia AU - Schwamborn, Jens C. AU - Jan, Jeng-Shiung AU - Deli, Mária Anna TI - A Triple Combination of Targeting Ligands Increases the Penetration of Nanoparticles across a Blood-Brain Barrier Culture Model JF - PHARMACEUTICS J2 - PHARMACEUTICS VL - 14 PY - 2022 IS - 1 PG - 19 SN - 1999-4923 DO - 10.3390/pharmaceutics14010086 UR - https://m2.mtmt.hu/api/publication/32575627 ID - 32575627 LA - English DB - MTMT ER - TY - JOUR AU - Ménesi, Dalma AU - Klement, Éva AU - Ferenc, Györgyi AU - Fehér, Attila TI - The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate JF - PLANTS-BASEL J2 - PLANTS-BASEL VL - 10 PY - 2021 IS - 10 PG - 15 SN - 2223-7747 DO - 10.3390/plants10102053 UR - https://m2.mtmt.hu/api/publication/32557484 ID - 32557484 N1 - Funding Agency and Grant Number: EU's Horizon 2020 research and innovation program [739593]; National Research, Development, and Innovation Office (NKFIH)National Research, Development & Innovation Office (NRDIO) - Hungary [K124828, K132486] Funding text: The project has received funding from the EU's Horizon 2020 research and innovation program under grant agreement No. 739593 and from the National Research, Development, and Innovation Office (NKFIH; grants K124828 and K132486). AB - Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test. LA - English DB - MTMT ER -