triloci-seq · Dechipering the triple helix code

Triloci-seq - Dechipering the triple helix code

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.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз