6 . 2021

Photoregulated oligonucleotide detachment as a way to improve the efficiency of polymerase chain reaction with reverse transcription for analysis at the single cell level

Abstract

Introduction. Due to the improvement of single cell RNA sequencing (scRNA-seq) technologies, a fundamentally new possibility to analyze the expression of groups of genes determining a specific function and allowing to trace the evolution of the cell both under normal conditions and during the development of pathology has appeared. Technologies, by means of which single cells are studied, make it possible to track mutational changes in genes of specific cell populations and find links with the manifestation of specific clinical features. Of greatest interest in this sense are lymphocyte populations and their receptors involved in antigen recognition. However, existing scRNA-seq technologies are not sufficiently effective for studying lymphocyte receptor repertoires. One of the limitations of the method is the insufficient efficiency of cDNA synthesis in the solid phase.

The aim of the study is to develop a technology for the synthesis and photoregulated release of oligonucleotides from the surface of a solid-phase carrier to increase the efficiency of cDNA synthesis from the mRNA of the а-chain T cell receptor gene of individual cells.

Material and methods. We developed a technology of oligonucleotide synthesis on a solid-phase carrier with the possibility of their release into a solution, if required. This process is provided by the incorporation of photosensitive molecules into the oligonucleotide structure at the synthesis stage. The linker is degraded under mild UV irradiation, which does not damage the biopolymer structure, resulting in release of the oligonucleotide sequence into the solution for the reverse transcription priming.

Results. A 39-nucleotide oligonucleotide sequence containing a short spacer, a photoactivated linker, a unique molecular barcode, a site for hybridization of the universal primer, and a fragment specific to the α-chain sequence of the T cell receptor were used. Synthesis was performed using «inverted» amidites in order to correctly orient the oligonucleotide on the carrier, ensuring cDNA synthesis. It was found that incorporation of the photosensitive PC-linker into the oligonucleotide structure, allows releasing all sequences available for UV-induced cleavage from the carrier surface within 5 min. Comparison of the production of the target product with and without photoactivation in the RT reactions showed significant differences in the amount of product obtained. The difference in the amount of cDNA in the two parallel reactions was approximately 7 PCR cycles, which suggests more than 100-fold excess of the initial amount of the specific target in the PC-linker reaction.

Conclusion. As a result of the study, we developed a technology for photoregulated oligonucleotide detachment from a solid carrier. We demonstrated that the PC-linker has no significant effect on the efficiency of polynucleotide chain synthesis and can be used in solid-phase synthesis of long deoxyribonucleotide sequences. Using emulsion PCR, we found that the use of a photodegradable linker significantly increases the efficiency of cDNA synthesis from single cell mRNA.

Keywords:solid-phase synthesis; photolysis; oligonucleotide release; PC-linker; cDNA; emulsion PCR

For citation: Kozlov I.B., Lebedin M.Yu., Gudima G.O., Sergeev I.V, Kofiadi I.A. Photoregulated oligonucleotide detachment as a way to improve the efficiency of polymerase chain reaction with reverse transcription for analysis at the single cell level. Immunologiya. 2021; 46 (6): 662-9. DOI: https://doi.org/10.33029/0206-4952-2021-42-6-662-669 (in Russian)

Funding. The study was supported by RFBR, project number 19-33-90076.

Conflict of interests. The authors declare no conflict of interests.

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