Long-run real-time PCR analysis of repetitive nuclear elements as a novel tool for DNA damage quantification in single cells: an approach validated on mouse oocytes and fibroblasts.

Since DNA damage is of great importance in various biological processes, its rate is frequently assessed both in research studies and in medical diagnostics. The most precise methods of quantifying DNA damage are based on real-time PCR. However, in the conventional version, they require a large amount of genetic material and therefore their usefulness is limited to multicellular samples. Here, we present a novel approach to long-run real-time PCR-based DNA-damage quantification (L1-LORD-Q), which consists in amplification of long interspersed nuclear elements (L1) and allows for analysis of single-cell genomes. The L1-LORD-Q was compared with alternative methods of measuring DNA breaks (Bioanalyzer system, γ-H2AX foci staining), which confirmed its accuracy. Furthermore, it was demonstrated that the L1-LORD-Q is sensitive enough to distinguish between different levels of UV-induced DNA damage. The method was validated on mouse oocytes and fibroblasts, but the general idea is universal and can be applied to various types of cells and species.

mRNA Vaccines: Why Is the Biology of Retroposition Ignored?

The major advantage of mRNA vaccines over more conventional approaches is their potential for rapid development and large-scale deployment in pandemic situations. In the current COVID-19 crisis, two mRNA COVID-19 vaccines have been conditionally approved and broadly applied, while others are still in clinical trials. However, there is no previous experience with the use of mRNA vaccines on a large scale in the general population. This warrants a careful evaluation of mRNA vaccine safety properties by considering all available knowledge about mRNA molecular biology and evolution. Here, I discuss the pervasive claim that mRNA-based vaccines cannot alter genomes. Surprisingly, this notion is widely stated in the mRNA vaccine literature but never supported by referencing any primary scientific papers that would specifically address this question. This discrepancy becomes even more puzzling if one considers previous work on the molecular and evolutionary aspects of retroposition in murine and human populations that clearly documents the frequent integration of mRNA molecules into genomes, including clinical contexts. By performing basic comparisons, I show that the sequence features of mRNA vaccines meet all known requirements for retroposition using L1 elements-the most abundant autonomously active retrotransposons in the human genome. In fact, many factors associated with mRNA vaccines increase the possibility of their L1-mediated retroposition. I conclude that is unfounded to a priori assume that mRNA-based therapeutics do not impact genomes and that the route to genome integration of vaccine mRNAs via endogenous L1 retroelements is easily conceivable. This implies that we urgently need experimental studies that would rigorously test for the potential retroposition of vaccine mRNAs. At present, the insertional mutagenesis safety of mRNA-based vaccines should be considered unresolved.

A comprehensive analysis of chimpanzee (Pan troglodytes)-specific LINE-1 retrotransposons.

Long interspersed element-1 (LINE-1 or L1) is one of the most abundant retrotransposons in the primate genomes and has contributed to their genome diversity and variations during the primate evolution. Among primate L1 subfamilies, L1Pt subfamilies include Pan troglodytes-specific L1s. L1Pt elements have been successfully expanded in the chimpanzee genome since the divergence of human and chimpanzee lineages. However, only a few full-length L1Pt copies were previously detected in the chimpanzee genome due to incomplete chimpanzee reference genome sequences at the time. In the present study, we aimed to identify chimpanzee-specific L1s using the most recent version of the chimpanzee reference genome (May 2016, panTro5). We identified a total of 3731 chimpanzee-specific L1s. This is much more than previously reported chimpanzee-specific L1 copies. Among these, 223 are full-length (>6 kb), and we annotated their subfamilies and examined their retrotransposition-competency. The result showed that there are two L1Pt subfamilies in the chimpanzee genome, and the nine structurally intact elements belong to L1Pt-1, L1Pt-2, and L1PA2 subfamilies. In addition, we found that the intact full-length L1 group showed significantly higher L1 expression level than the non-intact full-length L1 group using limited RNA-seq data. It is interesting to notice that the number of retrotransposition-competent elements is much less in the chimpanzee genome than that in the human genome. In conclusion, there is increasing evidence to indicate that chimpanzee-specific L1s have changed the chimpanzee genome, causing genomic difference from other primate genomes.