RNase H-based analysis of synthetic mRNA 5' cap incorporation.

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Reduced language processing asymmetry in non-autistic individuals with high levels of autism traits.

In the normal population the left hemisphere's predominance for language processing is well established. However, in disorders such as autism atypical patterns of hemispheric lateralisation are common. Given increasing recognition of the idea that autism represents a continuum, we investigated whether the reduced/absent hemispheric asymmetry for language processing extended into the normal population at the upper end of the autism spectrum. A total of 51 participants completed the AQ questionnaire (Baron-Cohen et al., 2001), and a lateralised lexical decision task assessing identification of concrete and abstract words. Based on the clinical finding of decreased hemispheric asymmetry in people with autism, we anticipated reduced hemispheric lateralisation in non-autistic people with higher levels of autism traits. Consistent with prediction, whereas people with lower AQ scores showed a clear right visual field (left hemisphere) advantage for word/nonword discrimination, people with higher AQ scores showed equivalent performance for the left and right visual fields. Our data indicate reduced left hemisphere language dominance in people with higher levels of autism traits, just as people with a clinical diagnosis of autism show atypical lateralisation. Moreover, the data offer further support for the notion that autism is a continuum, rather than a categorical diagnosis, with atypical patterns of hemispheric asymmetry being characteristic of people at the upper end of the spectrum.