Dependence Of NUcleosome Transactions on Sequence

Información del proyecto

DONUTS

Identificador del acuerdo de subvención: 101092623

DOI

10.3030/101092623

Fecha de la firma de la CE 14 Julio 2023

Fecha de inicio 1 Noviembre 2023

Fecha de finalización 31 Octubre 2028

Financiado con arreglo a

• European Research Council (ERC)

Coste total

€ 2 137 145,00

Aportación de la UE

€ 2 137 145,00

Coordinado por

UPPSALA UNIVERSITET
Sweden

Periodic Reporting for period 1 - DONUTS (Dependence Of NUcleosome Transactions on Sequence)

Período documentado: 2023-11-01 hasta 2026-04-30

Resumen del contexto y de los objetivos generales del proyecto

Living cells store their genetic information in DNA, but this DNA is not freely accessible. In eukaryotes, it is tightly packaged into a structure called chromatin, whose basic unit is the nucleosome: a segment of DNA wrapped around a set of proteins. While this packaging is essential for fitting DNA into the cell nucleus, it also limits access to genes. Cells therefore rely on specialised molecular machines, known as chromatin remodelling enzymes, to reposition nucleosomes and control when and where genes can be read, copied, or repaired.
A central but unresolved question in biology is how the DNA sequence itself influences the way nucleosomes are moved by these remodelling enzymes. Although it has long been known that DNA sequence matters, the molecular mechanisms underlying this sequence dependence remain poorly understood. One major reason is technical: existing experimental methods either provide detailed information on only a handful of DNA sequences at a time, or average over millions of molecules and cannot capture transient intermediate steps.
The overall objective of this project is to overcome this limitation by developing a new experimental platform that combines single-molecule measurements with modern DNA sequencing technology. This platform, called DONUTS (Dependence Of NUcleosome Transactions on Sequence), is designed to observe how individual molecules behave in real time while simultaneously identifying the DNA sequence involved. By doing so, the project aims to uncover how DNA sequence encodes rules for nucleosome motion and chromatin organisation.
In the long term, this knowledge is expected to deepen our understanding of gene regulation, genome stability, and epigenetic control, with relevance for fundamental biology and for diseases linked to chromatin misregulation.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

During the first phase of the project, work focused on establishing the core technology required to link single-molecule dynamics to DNA sequence at very large scale. This effort led to a major methodological breakthrough: the development of a platform called MUSCLE (MUltiplexed Single-molecule Characterization at the Library scalE), which implements the central concept of DONUTS in a robust and broadly applicable form.
MUSCLE enables thousands to tens of thousands of individual molecules to be observed in parallel on DNA sequencing chips, while preserving single-molecule resolution. Each molecule’s dynamic behaviour is recorded by fluorescence microscopy and then directly linked to its DNA sequence using standard sequencing technology. This makes it possible to explore how molecular behaviour changes across large libraries of DNA variants in a single experiment.
As a proof of principle, the platform was applied to study two different systems: the folding and unfolding of DNA structures, and the way the CRISPR-Cas9 gene-editing enzyme interacts with its DNA targets. These experiments revealed that even small changes in DNA sequence can lead to strikingly different dynamic behaviours, including unexpected intermediate states that would be invisible to conventional methods.
In parallel, the project more broadly established a complementary technique that allows biochemical reactions to be triggered locally and precisely using light. This capability is essential for studying energy-driven molecular processes and is now routinely used in the laboratory. Together, these achievements provide a solid foundation for extending the approach to nucleosomes and chromatin remodelling in the next phase of the project.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

This project has advanced the state of the art by removing a fundamental bottleneck in molecular biology: the inability to study complex molecular dynamics across large numbers of different DNA sequences at the single-molecule level. Before this work, researchers had to choose between detailed but low-throughput experiments on a few sequences, or high-throughput sequencing-based approaches that average over many molecules and miss transient behaviour.
The MUSCLE/DONUTS approach uniquely combines the strengths of both worlds. It allows researchers to observe individual molecules in action, detect short-lived intermediate states, and directly connect these behaviours to DNA sequence information across large libraries. This represents a conceptual shift in how sequence–function relationships can be explored at the molecular level.
While the platform was originally conceived to study nucleosome dynamics, its successful generalisation to other molecular systems was an important advance. It demonstrates that the method is broadly applicable to many DNA- and protein-based processes, from gene regulation to genome editing and beyond.
By enabling mechanistic studies at a scale previously accessible only to genomics, the project opens new possibilities for understanding how biological information is encoded and interpreted at the molecular level. This capability is expected to have lasting impact across biophysics, molecular biology, and genomics, and to inspire new experimental strategies well beyond the original scope of the project.