Developing molecular tools for probing and modulating genomic spatial adjacency

In addition to the vast information encoded in DNA sequence, the genome has physical features that are also essential for its function, including its organization in three-dimensional space. The development of high-throughput technology has greatly advanced our understanding of the spatial organization of the genome but has also raised more questions.

In this thesis, we developed molecular tools to address the remaining challenges regarding the interplay between genomic organization and function. By breaking down the subject from the global architecture of the genome into an ensemble of spatially adjacent chromatin segments, we came up with different methods covering various aspects.

We demonstrated in Paper I that global spatial information can be transferred in the format of DNA sequence encoding pairwise spatial proximity between two distinct molecular objects. We have shown that by growing network from pairwise relationship encoded in DNA sequence, spatial features at a global scale can be recovered. The results from this work highlighted the potential of using pairwise adjacency as a fundamental unit for recording the spatial organization of complex molecular systems. The high programmability and versatility of nucleic acids make them an ideal medium for encoding this information.

With the aim of studying the pairwise relationship between genomic DNA in cells, we devised a CRISPR-dCas9 system for different purposes by leveraging its high programmability for genome targeting. In Paper III, we have shown that the re-designed guide RNA can direct dCas9 to a pair of genomic loci, inducing DNA contacts. This system can be applied as a modulation tool to introduce pairwise contacts for decoding functional implications in cells. In Paper IV, we developed a method for the direct detection of pairwise interactions between genomic loci at the single-cell level in situ. This method is achieved by conjugating oligonucleotide tags to Cas9 and using the tags for probing the spatial adjacency between a pair of genomic loci targeted by Cas9.

Meanwhile, we developed an efficient method to fabricate and purify DNA origami with modifications in Paper II. This method makes the production of functionalized nanostructures more time and material-efficient compared to established techniques. The ease of production allows broader applications of functionalized nanostructures, including characterizing the effect of nanoscale distance on biochemical assays, as shown in Paper IV.

List of scientific papers

I. Hoffecker I. T, Yang Y., Bernardinelli G., Orponen P. & Högberg B. A computational framework for DNA sequencing microscopy. Proceedings of the National Academy of Sciences. 116, 19282–19287 (2019).
https://doi.org/10.1073/pnas.1821178116

II. Smyrlaki I., Shaw A., Yang Y., Shen B. & Högberg B. Solid Phase Synthesis of DNA Nanostructures in Heavy Liquid. Small. 19, 2204513 (2023).
https://doi.org/10.1002/smll.202204513

III. Yang, Y., Shen, B., Rocamonde L. I., Berzina I., Zipf J.& Högberg B. Reengineered guide RNA enables DNA loop and contacts formation in vivo. [Manuscript]

IV. Yang, Y., Rocamonde L. I., & Högberg, B. In situ detection of genomic cisinteraction by proximity ligation assay using oligonucleotide-labelled Cas9. [Manuscript]