High-resolution visualization and quantification of nucleic acid–based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS)

Abstract

Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

Metabolomics in Bone Research

“…To comprehensively identify metabolic pathways that are affected during bones diseases, we conducted an integrating enrichment and pathway topology analysis from the data of Table 1 by using Metabo Analyst 5.0 [ 94 ]. For this, we used the metabolites that are reported as significantly altered in osteoporosis, arthritis, and osteosarcoma.…”

Application of long-read sequencing to the detection of structural variants in human cancer genomes

“…This is because integrative analyses can be conducted, for example, DNA methylation, SVs, and haplotype information. It was reported that the accuracy of methylation calling from the long-read sequencing had high concordance with the short-read sequencing [78] . Therefore, DNA methylation analyses using long-read sequencing will flourish.…”

“…Our group has also developed a method combining Nanopore-type sequencing and Enzymatic Methyl-seq (EM-seq) [77] , named nanoEM [78] . EM-seq converts bases from unmethylated cytosines to uracils using an enzymatic reaction that prevents the fragmentation of DNA libraries during bisulfite sequencing, which is used to determine the pattern of DNA methylation.…”

Applications of Ionic Liquids in Whole-Cell and Isolated Enzyme Biocatalysis

“…Most of the enzyme structure-activity information available in the protein data bank ( accessed on 1 August 2021) or bioinformatics tools ( accessed on 1 August 2021) has been determined in aqueous media [ 50 , 51 ]. Enzyme structural stability in neat organic solvents or aqueous/organic biphasic media is commonly measured by activity assay.…”