At the core of molecular biology lies the intricate interplay between sequence, structure, and function. Single-molecule techniques provide in-depth dynamic insights into structure and function, but laborious assays impede functional screening of large sequence libraries. We introduce high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), integrating single-molecule fluorescence with next-generation sequencing. We applied SPARXS to study the sequence-dependent kinetics of the Holliday junction, a critical intermediate in homologous recombination. By examining the dynamics of millions of Holliday junctions, covering thousands of distinct sequences, we demonstrated the ability of SPARXS to uncover sequence patterns, evaluate sequence motifs, and construct thermodynamic models. SPARXS emerges as a versatile tool for untangling the mechanisms that underlie sequence-specific processes at the molecular scale.@en

Interactions are essential to life. At the cellular level, the most fundamental processes rely on interactions between DNA, RNA, and proteins. Given that the structure and function of these molecules are determined by their sequence, it is ultimately the sequence that dictates the strength and specificity of these interactions. Thus, to improve our understanding of biological processes, and even predict or manipulate them, it is imperative to obtain a comprehensive and quantitative overview of the relationship between sequence and interactions. One valuable method for studying these interactions in detail is single-molecule fluorescence microscopy. However, this technique is expensive and labor-intensive, making it impractical for large sequence libraries. In this thesis, we introduce a novel approach called SPARXS: Single-molecule Parallel Analysis for Rapid eXploration of Sequence space. By integrating single-molecule fluorescence microscopy with next-generation sequencing, this platform enables the simultaneous profiling of interactions with thousands of different DNA or RNA sequences at the single-molecule level.@en

Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon ‘Candidatus Harpocratesius repetitus’ shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs.

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Transposable elements have both detrimental and beneficial effects on their host genome. Tetrahymena is a unicellular eukaryote that deals with transposable elements in a unique way. It has a separate somatic and germline genome in two nuclei in a single cell. During sexual reproduction, a small RNA directed system compares the germline and somatic genome to identify transposable elements and related sequences. These are subsequently marked by heterochromatin and excised. In this Review, current knowledge of this system and the gaps therein are discussed. Additionally, the possibility to exploit the Tetrahymena machinery for genome editing and its advantages over the widely used CRISPR-Cas9 system will be explored. While the bacterial derived CRISPR-Cas9 has difficulty to access eukaryotic chromatin, Tetrahymena proteins are adept at acting in a chromatin context. Furthermore, Tetrahymena based gene therapy in humans might be a safer alternative to Cas9 because the latter can trigger an immune response.

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