Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source "Turkish DeLight".

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome.

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3'NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.

Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics.

The novel coronavirus pneumonia (COVID-19) is a highly infectious acute respiratory disease caused by Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (Prec Clin Med 2020;3:9-13, Lancet 2020;395:497-506, N. Engl J Med 2020a;382:1199-207, Nature 2020;579:270-3). SARS-CoV-2 surveillance is essential to controlling widespread transmission. However, there are several challenges associated with the diagnostic of the COVID-19 during the current outbreak (Liu and Li (2019), Nature 2020;579:265-9, N. Engl J Med 2020;382:727-33). Firstly, the high number of cases overwhelms diagnostic test capacity and proposes the need for a rapid solution for sample processing (Science 2018;360:444-8). Secondly, SARS-CoV-2 is closely related to other important coronavirus species and subspecies, so detection assays can give false-positive results if they are not efficiently specific to SARS-CoV-2. Thirdly, patients with suspected SARS-CoV-2 infection sometimes have a different respiratory viral infection or co-infections with SARS-CoV-2 and other respiratory viruses (MedRxiv 2020a;1-18). Confirmation of the COVID-19 is performed mainly by virus isolation followed by RT-PCR and sequencing (N. Engl J Med 2020;382:727-33, MedRxiv 2020a, Turkish J Biol 2020;44:192-202). The emergence and outbreak of the novel coronavirus highlighted the urgent need for new therapeutic technologies that are fast, precise, stable, easy to manufacture, and target-specific for surveillance and treatment. Molecular biology tools that include gene-editing approaches such as CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER and PAC-MAN, antisense oligonucleotides, antisense peptide nucleic acids, ribozymes, aptamers, and RNAi silencing approaches produced with cutting-edge scientific advances compared to conventional diagnostic or treatment methods could be vital in COVID-19 and other future outbreaks. Thus, in this review, we will discuss potent the molecular biology approaches that can revolutionize diagnostic of viral infections and therapies to fight COVID-19 in a highly specific, stable, and efficient way.