August 2020

Bio-Synthesis Newsletter - August 2020

CRISPR-Cas9 genome editing can cause deletions!

QDotRecently three research groups reported that the CRISPR-Cas9 causes deletions in the genome when used to induce double-strand breaks in human pluripotent stem cells (hPSCs). One study found that p53 inhibits CRISPR-Cas9 in hPSCs. A second study using long-read sequencing and long-range PCR genotyping for the study of DNA break repair mechanisms observed genomic damage in active cells. A third study reported a p53-mediated DNA damage response. More research appears to be needed to study the gene editing effects of CRISP-Cas9 in hPSCs. The use of bridged nucleic acids (BNAs) for CRISPR-Cas9 based gene editing may have the potential to improve selectivity of the system. 

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A pre-miR-21 binding cyclic peptide

QDotA cyclic peptide binds to the miR-21 precursors. Shortridge et al. identified a cyclic peptide as a ligand for the oncogenic microRNA-21 to overcome difficulties in targeting RNA secondary structures with small molecules. The research group recently developed a cyclic β-hairpin peptidomimetic which binds to the RNA stem-loop structure of the miRNA-21 precursor (pre-miR21) with strong affinity and specificity. The study screened a cyclic peptide library to discover the lead structure which bound to pre-miR21 with a KD = 200 nM and with preference over other pre-miRNAs. The NMR structure of the complex revealed that the peptide recognized the Dicer cleavage site. The cyclic peptide allowed altering the processing of the precursor to the mature miRNA in vitro and cultured cells. This study provided a rationale for the peptide binding activity and guidance for further improvements in affinity and targeting for cyclic peptides.

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Micro RNA can inhibit the expression of mRNAs!

Vitamin C[0]Zeng et al. in 2002 showed that both natural, as well as designed micro RNAs, inhibit expression of cognate mRNAs when expressed in human cells. Animal cells express a range of ~22 nucleotide noncoding RNAs called micro RNAs (miRNAs). The introduction of synthetic siRNA can induce the degradation of homologous mRNAs. In mammalian cells, induction of RNAi requires the transfection of RNA oligonucleotides. However, this process can be inefficient and also give rise to only transient inhibition of target gene expression. Authentic human miRNA, mir-30, was excised by Zeng et al. from an irrelevant mRNA transcript that contained the predicted 71 nucleotide mir-30 precursor molecule. The plasmid-encoded mir-30 miRNAs blocked translation of an indicator mRNA in human cells. According to Zeng et al., novel designed miRNAs can also be excised from mRNA transcripts containing synthetic miRNA precursor sequences. These artificial miRNAs can selectively inhibit the expression of mRNAs that possess a complementary target sequence.

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A new way to synthesize phosphorothioates

QDotRecently scientists developed a new synthesis approach using P(V)-based reagents for the programmable, traceless, and diastereoselective incorporation of phosphorus-sulfur nucleotides into oligonucleotides. Phosphorothiote nucleotides allow the synthesis of antisense oligonucleotides (ASOs) or of cyclic dinucleotides. A stereo-controlled incorporation of phosphorothioates into di-, tri-, or oligonucleotides is desired. At present, the reported yields for this new synthesis approach are not yet as high as for a P(III) reagent-based synthesis. However, P(III) based reagents are air and moisture sensitive and require special equipment to handle, whereas these new reagents are reported to be bench stable. However, further optimization of the coupling reactions will be needed to establish efficient commercial synthesis protocols.   

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