진학프로석·박사 채용 접수 플랫폼

KEY WORDS: RNA structure, thermodynamics of modified RNA, pseudouridine, N1-methylpseudouridine, chemical mapping of RNA structure

Principal Investigator: Prof. Ryszard Kierzek

Research topic: Effect of RNA modifications on the structure and functions of natural RNAs and in vitro transcribed vaccine mRNAs (IVT mRNA).

We offer the position of postdoctoral research assistant (post-doc) within the project 2022/45/B/ST4/03586 entitled "Thermodynamics of modified RNAs. The influence of RNA modifications on the structure and functions of natural RNAs and in vitro transcribed vaccine mRNAs (IVT mRNA)” funded by the National Science Center.

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2. Project description

RNAs are one of the most important biomolecules. In RNA, beside canonical nucleotides, are over 140 modified ones. The most abundant are N6-methyladenosine and pseudouridine. It is well established that different RNA biological functions are often related to their structure and for that reasons the knowledge about RNA structure is very important. In addition to many experimental systems of chemical mapping of RNA structure, there is also a method for predicting of RNA structure based on thermodynamic rules of RNA folding. Various programs are used for that prediction and RNAstructure is the leading one.

The aim of the research project is to determine full set of the thermodynamic parameters necessary to predict folding of RNAs containing the following modifications: pseudouridine (P), N1-methylpsuedouridine (1MeP), 5-methoxyuridine (5moU) and 5-methylcytidine (5meC). The collected thermodynamic parameters will be implemented into the RNAstructure program and allow to predict folding of natural RNAs (with modifications at selected positions) and vaccine type RNAs (with P, 1MeP, 5moU and 5meC replacing all uridines or cytidines, respectively). The next stage of the project will be the chemical mapping of several RNAs containing the chosen nucleotides and the comparison of RNA structures predicted with the modified RNAstructure program and determined experimentally with chemical mappings. The project is also important for reason related with SARS-CoV-2 virus pandemic. The most effective IVT mRNA vaccines from Pfizer and Moderna carrying spike mRNA in which all uridines were replaced with N1-methylpseudouridine. Earlier studies have shown that the introduction of N1-methylpseudouridine into IVT mRNAs most significantly increases mRNA expression as well as stability of mRNA in the cellular environment and indicated the best immunological respond. Other modified nucleotides that also showed very promising features were: pseudouridine, 5-methoxyuridine and 5-methylcytidine. This was also the reason why the particular modified RNA nucleotides were selected for investigations in this project.

The research plan includes the following steps:

(1) synthesis of necessary modified phosphoramidites and RNA oligonucleotides containing pseudouridine, N1-methylpsuedouridine, 5-methoxyuridine and 5-methylcytidine at specific positions,

(2) measurements of the thermodynamic stability of complementary duplexes and duplexes containing modifications within nonhelical RNA motifs. The project concerns determination of modified RNA thermodynamic parameters applicable to natural RNAs with modifications as well as to vaccine type RNAs. For each type of RNA modification, the next step will be calculation of respective thermodynamic parameters and theirs implementation into RNAstructure program,

(3) chemical mapping of two large fragments of the 28S subunit of the human ribosomal RNA (rRNA). Both model rRNAs contain 5 and 13 pseudouridine residues, respectively. They are selected from region of peptidyl transferase center (PTC) of rRNA. Based on the results of the chemical mapping, the RNAs secondary structure will be solved and compared with the structure predicted with the modified RNAstructure program. The comparison of both secondary structures will also serve for eventually improvement of the modified thermodynamic parameters and RNAstructure program,

(4) chemical mapping of two vaccine type RNAs. The selected RNAs are: subgenomic RNA M from SARS-CoV-2 virus (ca. 800 nt) and segment 4 mRNA from influenza A virus (ca. 1800 nt). For sgRNA M the chemical mapping will be performed on RNAs containing P, 1MeP, 5moU and 5meC, respectively. Also, segment 4 mRNA containing pseudouridine and N1-methylpseudouridine will be mapped. Next, their structures predicted with RNAstructure and determined based on chemical mapping will be compared,

(5) determination the structure of small RNA fragments containing modified pseudouridine and N1-methylpseudouridine by NMR and crystallographic methods. These studies are aimed at determining the interactions responsible for the significant stabilization of RNA structures by both modifications.

Overall, the project is important for understanding biological functions and the effects of modifications on RNA structure. The aspect of vaccine RNAs is extremely important also, as the effectiveness of the Pfizer and Moderna vaccines indicates that this is the right direction for the future development of IVT mRNA vaccines.