The synthesis of Broccoli RNA fluorescent aptamer in Saccharomyces cerevisiae yeast cells

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Abstract

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.

About the authors

Ousama Al Shanaa

Saint Petersburg State University

Author for correspondence.
Email: st072427@student.spbu.ru
ORCID iD: 0000-0003-1462-1687
SPIN-code: 7453-9258

Junior Research Associate of the Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg

Andrei M. Rumyantsev

Saint Petersburg State University

Email: rumyantsev-am@mail.ru
ORCID iD: 0000-0002-1744-3890
SPIN-code: 9335-1184
Scopus Author ID: 55370658800

Cand. Sci. (Biol.), Junior Research Associate of the Department of Genetics and Biotechnology
Russian Federation, Saint Petersburg

Elena V. Sambuk

Saint Petersburg State University

Email: e.sambuk@spbu.ru
ORCID iD: 0000-0003-0837-0498
SPIN-code: 8281-8020
Scopus Author ID: 6603061322

Dr. Sci. (Biol.), Professor of the Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg

Marina V. Padkina

Saint Petersburg State University

Email: m.padkina@spbu.ru
ORCID iD: 0000-0002-4051-4837
SPIN-code: 7709-0449
Scopus Author ID: 6602596755

Dr. Sci. (Biol.), Professor of the Department of Genetics and Biotechnology

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Diagram showing the design of the reporter system containing: tandem Broccoli aptamer, Hammerhead (HH) and Hepatitis Delta Virus (HDV) ribozymes, in addition to two restriction sites (HindIII and XhoI) (a); diagram showing the secondary structure of RNA transcript demonstrating the correct folding of the aptamer and the HH ribozyme facilitated by sequence complementarity added to the aptamer sequence (homology arms) (b)

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3. Fig. 2. Scheme showing the construction of the pYES2x2Broccoli plasmid. The genetic elements of the reporter construct. Hammerhead (HH) and Hepatitis Delta Virus (HDV) ribozymes in red, the tandem repeat of the Broccoli aptamer sequence in green; the necessary plasmid genetic elements for maintaining the vector in bacteria. Origin of replication (ori) and ampicillin resistance gene (AmpR) in grey; the necessary plasmid genetic elements for maintaining the vector in yeasts and for the expression of the reporter construct in blue, including: PGAL promoter, CYC terminator, selectable marker — URA3 gene, origin of replication 2μ ori

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4. Fig. 3. Electrophoregrams of the genetic elements of the expression cassette. a — GAL1 promoter gene in lane 1 was amplified using specific PGAL-F-BamHI and PGAL-R primers (530 bp); b — x2Broccoli tandem repeat (292 bp) in lane 1 was amplified using Broccoli 2F and Broccoli 2R primers; c — total expression cassette (1167 bp) in lane 2 including the reporter construct with GAL1 promoter, Broccoli aptamers, the pair of ribozymes HH and HDV and CYC1 terminator was amplified using PGAL-F and CYC1-R primers; d — restriction analysis of the pYES2x2Broccoli plasmid using BglI restriction enzyme in lane 3 (6168 bp) at two BglI restriction sites, one within the expression cassette construct and the other is not, resulting in two DNA fragments: 1510 bp and 4658 bp. 100 bp and 1kbp DNA ladders (Evrogen) were used as DNA size markers. All sizes of the amplified fragments correspond to the theoretically expected ones

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5. Fig. 4. Average fluorescence values (at a wavelength of 520 nm) of S. cerevisiae cell suspensions incubated in media with glucose and galactose, respectively. The measurements were carried out for 4 replicas. 95% CI is shown

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6. The sequence of the expression cassette in the pYES2x2Broccoli plasmid

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Copyright (c) 2022 Shanaa O.A., Rumyantsev A.M., Sambuk E.V., Padkina M.V.

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This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
 


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