Author: visnjevac

The fifth edition of the Hot Topics in Contemporary Crystallography advanced workshop (HTCC5), this time co-funded by Croatian Science Foundation, through the calixDNA project, and supported by European Crystallographic Association and International Union of Crystallography, was held in Sheraton Dubrovnik Riviera Conference centre in Mlini, from 16^th to 21^st April 2023. The workshop is tradionally organized be the Croatian Association of Crystallography, and chair of the international Organizing committee was dr. Aleksandar Višnjevac, head of the calixDNA project. The HTCC5 was generously sponsored by Bruker, Formulatrix, Douglas Instruments, Xtal Concepts, Anton Paar, Mitegen, Oxford Cryosystems, Dectris and Rigaku.

The workshop gathered 52 registered participants from 16 countries (Algeria, Austria, Croatia, Czech Republic, France, Germany, Italy, Ireland, Jordan, Poland, Serbia, Sweden, Slovenia, Turkey, UK, and Ukraine) out of which 28 students, 11 lecturers, 7 sponsor representatives, 2 accompanying persons and 4 organizers. 13 students (from Algeria, Austria, Czech Republic, Poland, Croatia, Serbia, Sweden and Ukraine) received bursaries to cover, partially or entirely, their registration fees, accommodation expenses and travel expenses. The bursaries were generously funded by International Union of Crystallography, Croatian Association of Crystallographers and European Crystallographic Association.

Over four intensive working days, two of which extended to 11:00 PM, four “hot topics” (macromolecular crystallization, processing of the diffraction data, structural databases and visualization of the macromolecules) were thoroughly covered through 15 lectures, 14 tutorials and one demonstration/presentation. In addition to this, the Douglas Instruments and Xtal Concepts Activity “Bring your own sample” enabled students the touch-on experience in applying advanced techniques (dynamic light scattering, microseeding) on their own brought samples with a goal (that, in quite a few cases, was indeed achieved) to produce high quality single crystals, suitable for diffraction measurements. Finally, within the interactivity scheme of the HTCC5, students, themselves, presented 18 posters and 15 five-minutes long discussion triggering presentations (DTP). THe best presentations were awarded with a monetary 100 € award which was generously provided by Dectris (best DTP) and Croatian Association of Crystallographers (best poster). he best poster prize was awarded to Daniel Mokos, for his poster entitled “Crystal structure and proposed mechanism of A622 homologues from the nicotine biosynthesis pathway”, while Marika Majerova was awarded for the best discussion triggering presentation for her presentation entitled “An extant enzyme with dual dehalogenase‒luciferase function”.

Dr. Aleksandar Višnjevac spent a month in the partner lab at the University of Paris Cite with the task of synthesizing gram quantities of SBB1/SBB2 which have been identified as ideal candidates for the recognition of various DNA/RNA chains. The product (SBB1) is successfully prepared and we are all set now for the second round of crystallization experiments with the macromolecular complexes formed by SBB1, Cu(I) complex of SBB1 and SBB2 and chosen polynucleic chains.

Within the frame of the educational activities envisaged within the calixDNA project, Aleksandar Višnjevac has attended highly renowned Cold Spring Harbor Laboratory (CSHL) Macromolecular Crystallography course, from Oct, 9^th to 25^th, 2022. During over 220 working hours, participants are acquainted, by means of the hands-on individual experience, with the entire process of macromolecular crystal structure determination, from the protein crystallization to data collection, data processing, validation and deposition.

Refined structure (R_free = 0.1605) of the Proteinase K from Tritirachium Album crystallized, solved and refined in the frame of the CSHL mmCourse by A. Višnjevac

Ivana Nikšić-Franjić is, from Oct. 3^rd to 7^th, 2022, at the Institute for Molecular Biosciences of the University of Graz (group of prof. Karl Gruber) performing first set of over 2000 crystallization experiments within the frame of calixDNA project. Ivana is searching for initial crystallization conditions of the macromolecular [SBB4/d(GTATATATAC)₂] complex. This research visit is realized by courtesy of prof. Karl Gruber, scientific advisor of the calixDNA project, which is deeply acknowledged. This Ivana’s visit to Graz is a major step forward for the calixDNA project, as it marks the kick-off of the activities in the domain of macromolecular crystallography which is envisaged to be a cornerstone of the entire project. Fingers crossed.

On 13^th of July, 2022, from 11:00 to 12:20 CET, the fifth meeting of the calixDNA research team was held, in presence of A. Višnjevac, I. Piantanida, O. Reinaud, B. Kojić-Prodić, K. Gruber, I. Nikšić-Franjić, D. Pavlović Saftić, M. Wallet and M. Legras. M. Wallet and M. Legras presented the results obtained during their internship at the calixDNA project, which started on April 23^rd, 2022, and will continue until the end of July 2022, which was followed by the vivid discussion and many questions to the presenters. Research stay of I.Nikšić-Franjić at the research group of prof. K. Gruber was confirmed for the late September/early October, during which she will set up the first round of crystallization experiments of the genZ SBB4 – DNA oligomere complexes. Research visits of A. Višnjevac and I. Nikšić Franjić to the Paris lab, where they will continue the synthesis of the gram quantities of the most promissing SBB derivatives, was also mentioned and confirmed.

The presentation is available here.

Board of Directors of the Croatian Science Foundation, at its 27th session held on June 17^th, 2022, based on the evaluations and recommendations of the evaluators, made a decision (Class: 120-02/22-02/13, Registration number: 63-02/ 01-22-33) on the acceptance of the 1st periodic report of calixDNA project (Feb. 1^st, 2021 – Jan. 31^st, 2022). Final grade B (Good progress) has been assigned to the report with the remark that “the project has achieved most of the goals for the given period, and the reasons for not achieving some goals are justified”. Consequently, the board has decided to continue financing the project.

calixDNA project was presented to the widest audience in Croatia by the well known science&technology dedicated TV show “Prometej” which was broadcasted on 4^th od June, 2022 at 2:30 PM at HRT1. The cameras of the HRT entered the Laboratory of Chemical and Biological Crystallograhy and Laboratory for biomolecular interactions and spectroscopy at the Ruđer Bošković Institute to get an insight into the research process within the calixDNA project. The project was presented by its head, dr. sc. Aleksandar Višnjevac, and particular research activities by dr. sc. Ivana Nikšić-Franjić, Malorie Wallet and Marine Legrass, team members.

calixDNA team is joined, from April 23^rd, 2022, by Marine Legras and Malorie Wallet, students of the second year of chemistry at the ENSICAEN (Ecole Nationale Superieure de Caen). During their internship at the Ruđer Bošković Institute, they will be working on the recognition and binding studies of the gen1 supramolecular biomimetic binders, as well as on the series of crystallization experiments pertaining to the structural studies of the genZ SBB- mononulceotide tweezer like complexes, and metal complexes of some of the genZ SBBs. Both Malorie and Marine will stay with us for a period of three months.

Upon invitation by the editorial board of New Journal of Chemistry, our artwork illustrating recently published paper Impact of positive charge and ring-size on the interactions of calixarenes with DNA, RNA and nucleotides, will appear on the frontpage of this journal (NJC, Issue 15, online 11/04/2022). The illustration was prepared by authors I. Piantanida and A. Višnjevac and designed by Toni Lijić. Toni Lijić is a M.Sc in general chemistry and digital designer with a growing portfolio in natural sciences related illustrations. His signature stands also on the banner of calixDNA.org. For more on Toni’s work, please follow this link.

Our article entitled Impact of positive charge and ring-size on interactions of calixarenes with DNA, RNA and nucleotides by Ivona Krošl, Ena Otković, Ivana Nikšić-Franjić, Benoit Colasson, Olivia Reinaud, Aleksandar Višnjevac and Ivo Piantanida, has been accepted for publication in New Journal of Chemistry. The publication summarizes part of the work accomplished during the first product period, on the recognition and binding study of the selected SBVs towards variety of polynucleotide chains and some mononucleotides.

ABSTRACT

Comparison of various calix[6]arene and calix[4]arene derivatives revealed that only analogues bearing permanent positive charge non-covalently bind to ds-DNA and ds-RNA, by insertion into DNA minor groove or RNA major groove. Also, these cationic analogues revealed strong and highly selective charge-dependent stabilization of AT-DNA against thermal denaturation, calix[6]arene trication being for an order of magnitude more efficient than its calix[4]arene dicationic analogue. At variance to DNA/RNA selectivity for only cationic calixarenes, both, neutral and cationic calixarenes bind nucleoside monophosphates with similar efficiency, by forming tweezer-like supramolecular complexes, with nucleobases inserted between aromatic pendant arms grafted to calixarene rims. Such nucleotide-calixarene complexes were monitored by emission change of calixarene as a function of nucleobase insertion, at variance to DNA/RNA complexes in which calixarene is inserted into polynucleotide groove, which does not change calixarene emission – stressing importance of the ligand insertion within calix-basket for the fluorimetric sensing.