May 2019

Bio-Synthesis Newsletter - May 2019

Circular RNA and Innate Immunity

cirDNATo avid infectious agents and pathogens, mammals and insects have evolved an immune system able to control and eliminate pathogens. In mammals, including humans, the innate immune system is the first line of defense against invading pathogens. However, it is not at all clear how the innate immune system differentiates between self and non-self in terms of species-specific molecular patterns.Recent studiesindicate that non-self RNAs may be detected as naked RNAs possessing secondary and tertiary structures such as circular RNA . In vivo, eukaryotic RNAs are found to be less structured because of the presence of helicases and m6A epigenetic modificationsunfolding RNAs.
The human RNA-activated protein kinase, a dsRNA binding protein, recognizes viral and bacterial pathogens by binding to their RNA, is now known as an essential sensor in the innate immune response. Protein kinase R (PKR) is a major regulator of central cellular processes including mRNA translation, transcriptional control, the regulation of apoptosis, and cell proliferation.PKR was first identified as a cellular protector against viral infections. When activated, PKR phosphorylates the eukaryotic initiation factor eIF2α resulting inthe termination of translation.
The study of innate immune system involves the design of RNAs, PCR primers, hemiduplex templates, RNA preparation and purification, protein expression and purification, PKR activation assays, structure mapping, PKR footprinting, and RNA sequencing. As a side note: Circular caged oligonucleotides containing modified nucleotides such as bridged nucleic acids as well as morpholinos allow manipulation of gene expression in cells which maybe an additional avenue for the study of the innate immune system.

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Autism and vasopressin

VasopressinAutism, or autistic spectrum disorder (ASD), is a developmental disorder of variable severity characterized by difficulty in social interaction and communication as well as by restricted or repetitive patterns of thought and behavior. People with autism tend to have low levels of vasopressin in their cerebrospinal fluid. Presently, there are no medications for the treatment of these social behavior deficits. However, preclinical research suggests that the neuropeptide arginine vasopressin may offer a possible treatment for ASD.
The nine-amino-acid hormone vasopressin is synthesized in the brain and has many functions in the human body. To find out if vasopressin is useful for the treatment of ASD, Parker et al. in 2019 tested the efficacy and tolerability of a 4-week intranasal arginine vasopressin daily treatment in 30 children with ASD. In this study , mostly boys ages 6 to 12, were treated for four weeks with nasally delivered vasopressin. This way of delivery is thought to improve the chances of the hormone getting into the brain. Their preliminary findings suggest that arginine vasopressin delivered through the nasal cavity has the potential for treating social impairments in children with this disorder.

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Errors in next-generation sequencing

SSUnfortunately, next-generation sequencing (NGS) based deep-sequencing is not free of errors. Errors introduced during sequencing when they occur at the single nucleotide level can lead to misinterpretation of mutation rates, for example, when used for the detection of mutation rates in cancer tissue. Errors in NGS can occur at various steps during the current NGS workflows.
Sample handling, library preparation, PCR enrichment, as well as the process of sequencing itself, has the potential to introduce errors. Therefore, Ma et al. recently used the current NGS technology to evaluate the distribution of read-specific sequencing errors systematically. The research group discovered that the substitution error rate could be computationally suppressed 10−5 to 10−4, or 10- to 100-fold lower than generally considered achievable (10−3) in the current literature. The substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets were quantified. The observed error rates differed by nucleotide substitution types, ranging from 10−5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10−4 for A>G/T>C changes. C>T/G>A errors exhibit strong sequence context dependency. However, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. Luckily, more than 70% of hotspot variants can be detected at a 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. To conclude, further improvements in NGS analysis are needed to enhance the precision of deep sequencing and to minimize errors in sequence read-outs.

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CRISPR-based nucleotide editor randomly changes RNA

Crispr CasRecently Huang et al. combined SpCas9-NG with ABEmax to generate a new base editor called ABEmax-NG and demonstrated that ABEmax-NG performed precise A•T to G•C conversion and also coveredmore splicing sites. However, this CRISPR-based gene editing is not as precise as originally thought.
Recently scientist observed thatthe base editor designed to modify specific adenosines out of DNA also randomly modifies adenosines in RNA. However, it is not clear yet if this causes any biological effects since the turnover of RNA in cell is very high. To avoid off-target editing, the base editor may need to be redesigned.

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