Filip Bošković
Single-molecule, single-cell native transcriptomics with nanopores and RNA origami codes
Keywords: RNA origami, designed RNA codes, nanopore microscopy, native target transcriptomics.
In my thesis, I implemented physical approaches to studying biological systems using DNA/RNA origami technology and nanopore microscopy.
DNA origami is a self-assembly technique, where DNA is used for building, rather than for storing genetic information. It employs long single-stranded DNA molecules as scaffolds, which are folded into specific shapes using short, complementary DNA sequences. The resulting DNA nanostructures have a wide range of potential applications in biotechnology, including drug delivery, molecular sensing, and data storage. RNA origami is a similar technique that uses RNA molecules instead of DNA to fold into precise three-dimensional shapes.
RNA is an omnipresent molecule in living systems that governs numerous functions in the cell. It has also found a critical role in RNA vaccines and therapeutics. Herein, we reshaped RNA to designed RNA codes(RNA:DNA duplexes) by using complementary DNA sequences. With subnanometer precision, I placed structures that enabled their identification with nanopore microscopy. Nanopores are tiny holes in a membrane or a solid-state material that are only a few nanometers in diameter. When a molecule passes through the nanopore, it creates a change in the electrical current (some of the ionic current is blocked). Such changes can be measured and used to identify the different RNA codes.
In my PhD I employed these RNA and DNA codes to identify and study native RNAs [3]. Studying the native RNA diversity without the use of amplification or enzymes is important because it allows for a more accurate and representative analysis of RNA molecules in their natural state. Amplification methods such as PCR introduce biases and artifacts that can distort the original RNA profile, leading to incorrect conclusions about gene expression levels, alternative splicing events, and RNA modifications. In addition, enzymatic treatments, such as reverse transcription, can alter RNA integrity and introduce technical variability, making it difficult to distinguish between genuine biological signals and experimental noise. By studying the native RNA transcriptome, we can avoid these potential pitfalls and obtain a more reliable and comprehensive understanding of the RNA landscape in different cell types, tissues, development stages, and disease states.
We demonstrated native target transcriptomics in human cervical cancer total RNA by making RNA codes for different RNA structural variants, or isoforms, for genes of interest. Multiplexed identification is enabled by our RNA codes. We designed 10 unique current structural (pseudo)colours enabling the assembly of 10 billion RNA codes i.e., 10 billion RNA molecules simultaneously.
The results have profound importance in studying gene expression in complex biological samples, diagnostics of viruses and even their viral variants in patient swabs, and even the fundamental understanding of molecular transport. It paved the way for studying native ‘RNA makeup’ including RNA quantity, structure, dynamics, and their RNA-protein interactions at the single-molecule, single-cell level.
The cornerstone was to implement quantitative, physical approaches to understanding biological systems. My PhD was a concoction of physics and mathematics (nanopore electrical systems, guided origami, barcoding) aimed towards understanding RNA. Herein, I employed RNA codes made by DNA/RNA origami to identify native RNA transcripts. It enabled multiplexed targeting of up to 10 billion RNAs in parallel without need for prior amplification (PCR) or enzymes. This paves the way towards native single-cell transcriptomics.
My method was implemented in many projects, spanning from chemistry to physics and computation modelling, with various biological applications including tandem repeat disorder and infectious disease diagnostics.
Filip's publications
Cavendish Laboratory
Prof Ulrich F. Keyser Lab
Figure legend: A method has been developed to identify RNA transcript isoforms at the single-molecule level using nanopore microscopy. Here, target RNA is refolded into RNA codes with designed sets of short, complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colours named RNA codes. RNA codes are electrically readout using nanopore microscopy. RNA codes enable the identification and quantification of up to 10 billion RNAs in native transcriptome.
