RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

BACKGROUND: RNA aptamers are short, single-stranded oligonucleotides, with remarkable binding ability to target molecules characterized by high specificity and affinity. Such targets are vastly diverse and range from specific ions to entire cells. RNA aptamers are widely used in biology and medicine for basic research, as well as for practical purposes as in therapy and diagnostics. At present, chemical or in vitro methods of synthesis are mainly used to obtain RNA aptamers. However, such methods are expensive and time-consuming with low productivity. Therefore, in vivo methods are becoming more attractive to researchers working on optimizing high-scale production of RNA aptamers. AIM: The aim of this work is to develop a reporter system for optimizing the synthesis of small RNA molecules in Saccharomyces cerevisiae yeast cells. MATERIALS AND METHODS: We used the Broccoli fluorescent RNA aptamer to develop a reporter system allowing us to optimize the conditions for in vivo short RNA synthesis in yeast cells. This aptamer is about 112 bp in size and binds to the fluorogenic dye DFHBI-1T. Only upon binding, the aptamer-dye complex exhibits fluorescence properties. After excitation using light with a wavelength of 482 nm, the aptamer-dye complex emission is observed with a peak at 505 nm. RESULTS: We have designed a reporter system providing the synthesis of the fluorescent Broccoli RNA aptamer in S. cerevisiae yeast cells. Transcription of RNA molecules containing the aptamer is carried out by the regulated promoter of the GAL1 gene. The synthesized transcripts contain the HH and HDV ribozymes to ensure precise cleavage of the RNA aptamer sequences. CONCLUSIONS: This reporter system is based on the Broccoli RNA aptamer, and it can be used to optimize the in vivo synthesis of RNA aptamers in S. cerevisiae yeast cells. This work serves an urgent task in connection with the active use of such aptamers in scientific research, biotechnology and medicine.

Methylotrophic yeast Komagataella рhaffii (also known as Pichia pastoris) is widely applied in biotechnology for recombinant protein production. K. рhaffii particularly proved to be a successful host system for the synthesis of immunomodulators such as interferons [1]. In this study, we engineered K. рhaffii strains capable of producing the immunomodulatory protein Neoleukin (Neo-2/15). Neo-2/15 is an interleukin-2 mimetic, designed by in silico methods [2]. In preclinical studies on murine cancer models, Neo-2/15 showed superior therapeutic effect to inerleukin-2 with reduced toxicity. In this work, we show that K. рhaffii can successfully synthesize and secrete Neo-2/15. We have obtained a number of K. phaffii strains, including Mut^S and Mut⁺, with different Neo-2/15 expression cassettes integrated into the genome, carrying up to five copies of Neo-2/15 gene. In fact, the higher number of Neo-2/15 gene copies in K. рhaffii genome allowed a higher protein yield. In this study, we further developed a split marker approach [3] for yeast transformation using two DNA fragments, comprising of the expression cassette and the overlapping fragments of the marker gene. This allowed us to generate Mut^S strains with two copies using pPICZαB vector, which is not originally intended for Mut^S strain generation. As a result, we demonstrated that K. phaffii is a perspective producer of Neo-2/15, providing wide opportunities to increase the production of this therapeutic protein.

Background. A selective system based on the M1 virus of the yeast Saccharomyces cerevisiae was proposed. Methods. To create a recipient strain, a DNA fragment encoding the killer toxin of the M1 virus under the control of the regulated promoter of the GAL1 gene was inserted into the genome of S. cerevisiae strains Y-1236 and Y-2177. Results. Integration of such expression cassette leads to the conditional lethality resulting strains die on a medium with galactose when killer toxin synthesis occurs. A linear DNA fragment containing the gene of interest flanked by sequences homologous to the promoter of the GAL1 gene and the termination region of the CYC1 gene is used to transform the obtained strains. During transformation due to homologous recombination, the sequence encoding the killer toxin is cleaved and the transformants grow on a medium with galactose. Conclusion. The proposed selective system combines the main advantages of other systems: the use of simple media, without the need to add expensive antibiotics, and a simplified technique for constructing expression cassettes and selecting transformants.