Periodic Reporting for period 2 - triloci-seq (Triloci-seq - Dechipering the triple helix code) Reporting period: 2021-08-01 to 2022-07-31 Summary of the context and overall objectives of the project While the abundance of studies in the field of triplex formation is vast, direct evidence for triple helices in vivo is circumstantial. In particular, even after 60 years of research, there seems to be a lack of understanding of the underlying ‘triplex code’ in vivo. In other words, what constitutes the necessary sequence determinants that encode a high affinity (RNA) triplex forming oligo (TFO) and its triplex target site (TTS) are only qualitatively understood at the present time. Finally, yet importantly, the conflicting data and doubt in the lncRNA field (personal correspondence) of RNA-TFOs/lncRNAs potentially being able to target genomic loci within the chromatin context continues to puzzle researchers to this day. In this proposal, we hypothesized that in order to make significant progress on these questions, a new high-throughput deep- sequencing approach for studying triplex formation in vivo must be devised.To prove or disprove our hypothesis we devised a research plan containing in vitro, in cell, and synthetic biology components. The in vitro experiments were designed to help us map out the sequence space of possible triplex interactions in laboratory conditions. The goal here was to find sequence motifs, and not just individual sequences. The goal of the in cell experiments was to search whether the motifs discovered in vitro are also enriched in cell, thus providing strong evidence that indeed a triple interaction was taking place. Finally, the synthetic biology experiments were designed to validate the in cell and in vitro high throughput experiments, by showing that these triplex forming motifs can then be used as a deterministic biotechnological tool to control expression. The validation of triplex regulatory mechanism also serves a technological purpose, allowing us to develop a novel form of targeted gene-editing technology which can be used to target rare genetic diseases. Identification and characterization of a new programmable triplex-based regulatory mechanism has a vast potential for application in the biotechnological and personalized medicine field. For instance, it is hypothesized that some genetic disordered (e.g. Friedrich Ataxia) are caused by malfunction of the triplex mechanism in some genomic sequences, opening the door for a simple therapeutic solution that will alleviate these conditions. For the duration of the proposal, we have made substantial progress in this goals completing all proposed in vitro and in cell work. Triplex based formation for both single stranded RNA and DNA was characterized using novel high-throughput assays developed specifically for these tasks. Our in cell, showing that in vitro motifs are also found in cell, strongly support in cell triplex formation. Finally, validation of these finding with synthetic biology experiments is at the present time an ongoing process, with very promising preliminary results. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The work performed during the proposal has achieved several goals (most of which is yet unpublished). Full characterization for both ssRNA-dsDNA and ssDNA-dsDNA triplex formation in vitro using a novel click-based assay, which has allowed us for the first time make a comprehensive comparison of the two types of triplex reactions against a large library of identical target sites. This development breaks radically from existing state-of-the-art where only a handful of such complexes were characterized in such a detailed quantitative fashion. Our data shows that there is a substantial difference in the triplex forming oligos between RNA and DNA, when binding them to the same triplex target site library set. In particular, we are observing two populations: a G-rich correlated population that seems to indicate a preference to triplex formation of G-rich target sites. This was fairly well understood previously, and also reported by us in a previous publication. The second correlation is highly enriched for RNA-triplex populations and is poorly correlated to the DNA-triplex population. This population was previously unreported and thus constitutes a new type of triplex-forming population identified int his study. At the present time, we are still analyzing the sequences that have emerged from this population, and we have initial evidence that these sequences are relevant to in vivo settings.A second achievement of this proposal is that we were successful in showing that triplex-based transcriptional regulation can be used to induce transcription in vivo. We have been able to show both activation of transcription with synthetic long non-coding RNA containing a specific GAA-rich triplex forming motif, and in addition silencing of transcription using a ssDNA oligo containing the same GGA-rich motif. Both acted only on specific GAA-rich target sites that were previously characterized in vitro. At the present time we are not considering active forms of exploitations vis-a-vis writing a patent or commercializing the technology. This, however, may change if further data analysis will reveal exploitation opportunities. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) By the end of the project I expect to develop a triplex algorithm which will be able to score any 20-25 bp dsDNA sequence for its potential for triplex formation (i.e. Target site), and also to find its matching set of triplex forming oligo. I hope to validate the algorithm both in vivo and in vitro. Such an algorithm can the be used to score the triplex forming potential of naturally occurring long non coding RNA, and also point out their potential triplex target sites on the genome. These insights can then be further used to try to get a better understanding on some rare genetic diseases (e.g. Friedrich Ataxia) or some cancer-types, where triplex formation may play a role. In such case, developing drugs in siRNA format which either facilitate triplex formation in cases where it is deleted, or alleviating disease-associated triplexes may prove to be viable drug candidates for some incurable diseases. image-for-eu.jpg
