This year, Croatian Science Foundation, through the project calixDNA, in a frame of its strategic orientation towards the support of outreach and education activities, generously supports the organization of the advanced macromolecular crystallography workshop HTCC6, to be held in Dubrovnik, 7 – 12 April, with participation of over 50 students, bursarees, lecturers and tutors. The HTCC6 Programme book just came out of the printing office. At its very first page, the support of the HrZZ is acknowledged.

At its 66th session, held on 27^th Oct, 2023, the board of governors of the Croatian Science Foundation fully accepted the second periodic report of the calixDNA project (for the period from 1^st Feb. 2022 to 31^st July 2023), submitted earlier this year by the principal investigator. The Board recommended further funding of the project as planned, and allowed, following the proposal submitted by the P.I., the transfer of all unspent funds to the project budget for the upcoming, third (and last) project period. This decision of the Board will ensure normal functioning of the project for the remaining period, and is in line with the results obtained so far. The P.I. and the team members greatly appreciate positive reviews given by the independent evaluators and the decision by the Board of governors.

Our second publication Novel Pyrene-Calix[4]arene Derivatives as Highly Sensitive Sensors for Nucleotides, DNA and RNA, by Ivana Nikšić-Franjić, Benoit Colasson, Olivia Reinaud, Aleksandar Višnjevac, Ivo Piantanida and Dijana Pavlović Saftić, has just been accepted for publication in RSC Advances (Q2, IF = 3.9). The paper describes the recognition and binding studies of two newly prepared pyrene-calix[4]arene conjugates towards selected mononucleotides and polynucleic chains. It has been reported that our compunds display a remarkable affinity towards mononucleotides and are comparable with some of the most efficient supramolecular sensors for mononucleotides. They also bind to a ds DNA chain with an amazing efficiency.

The calixDNA project members and associates (Aleksandar Višnjevac, Olivia Reinaud, Ivo Piantanida, Ivana Nikšić-Franjić and Dijana Saftić had a brief online project meeting at 6th of July, 2023, from 9:30 to 10:20, to discuss current status of the research, in particular ongoing crystallization experiments and the forthcoming submission of the manuscript Pyrene-Calix[4]arene Derivatives as Highly Sensitive Sensors for Cations, Nucleotides, DNA and RNA which summarized the results on the binding and recognition studies of selected gen1 (rationally modified) supramolecular biomimetic binders (SBBs). A. Višnjevac gave an overview of the ongoing crystallization experiments, which was followed by a discussion, where O. Reinaud raised the issue of pH in the experiments involving the imidazole containing SBBs. I. Piantanida reported on the manuscript status and announced that the submission will happen shortly.

Dr. Ivana Nikšić-Franjić presented the concept, underlying ideas and results so far obtained within the frame of calixDNA project research activities at the Colloquium of the Division of organic chemistry and biochemistry of the Ruđer Bošković Institute, on June 6^th, 2023, upon invitation of the Colloquium coordinator, prof. dr. Nikola Basarić. The presentation was the part of our continuous efforts to disseminate activities of the project among the scientific community as well as in general public. It is, in our view, of a crucial interest for the successful continuation of the research activities at such an intrinsically multidisciplinary project as calixDNA, to maintain a vivid virtual platform for ideas exchange and communication among the scientists from the different disciplines.

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.

Roll of honor of the CSHL MAcromolecular Crystallography course

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.