Imagine you find a very nice cooking recipe online and you want to replicate it. You print it out and head to the kitchen. But when you get there, you realize that some of the steps have been printed several times. You begin to prepare the dish, but the repeated steps make it hard to follow – in some cases, you end up adding too much of one ingredient, and you ruin the dish. The instructions were wrong, your printer used more energy and ink than it could have, and you ended up having a terrible dish. Something similar can happen in a living cell. Deoxyribonucleic acid (DNA) is an organic molecule that contains the genetic information of an organism in a cell. DNA is made from nucleotides whose sequence define the genetic information stored. However, there are cases in which some nucleotide sequences in a given gene are unexpectedly repeated, which can cause a variety of issues, as we have revised in our cooking example. The production of unexpected expansions of nucleotide repeats, or tandem repeats, consume energy and resources of the cells. In some cases, it can affect the amino acid sequence of synthetized proteins, or the mRNA encoded with these repeats cannot be translated and can become entangled with functional proteins. All these processes can result in medical conditions broadly referred to as tandem repeat disorders. One type of tandem disease is myotonic dystrophy 1 (DM1), which arises due to the expansion of the CTG trinucleotide. The midi-project aims to explore a potential low-cost diagnosis method of DM1 using solid-state nanopore sensing of RNA-DNA hybrids to offer an alternative to conventional procedures, which are expensive and time consuming. Nanopore sensing is a single molecule detection method which analyses variations in an ionic current going through a nanopore. As molecules pass through the pore, ions are depleted, causing the current to drop. Larger structures produce larger current drops. mRNA encoded with the repeats can be hybridized with short oligos to which structural units, such as enzymes or DNA
origami structures, could be bounded to the regions with the repeats [1]. Drops in current at nanopore measurements could be associated to the repeats in the initial gene. A variety of data analysis methods are to be used to understand the nanopore signals detected and experiments can be designed to gain further understanding of nucleic acids and enzyme biophysics. Furthermore, we hope that RNA-DNA hybrid studies broaden the information that can be obtained from a given sequence in a short amount of time, such as isoform identification and genetic variations. These studies could be used to detect RNA molecules in single cells to develop a single-cell transcriptomics platform, and address questions related to stochastic nature of gene expression or to reveal cell heterogeneity in a cell population [2].
Relevant literature:
1. Boskovic, F. N., & Keyser, U. F. (2021). Nanopore microscope identifies RNA isoforms with structural colors. bioRxiv.
2. Kulkarni, A., Anderson, A. G., Merullo, D. P., & Konopka, G. (2019). Beyond bulk: a review of single cell transcriptomics methodologies and applications. Current opinion in biotechnology, 58, 129-136.
Image taken from:
Cuffari, B.(2017) RNA Delivery Mechanism for Disease Treatment and Vaccine Delivery. https://www.azonano.com/article.aspx?ArticleID=4638
NanoDTC PhD Student, c2021