Triloci-seq - Dechipering the triple helix code | triloci-seq | Proyecto | Ficha informativa | H2020 | CORDIS

Descripción del proyecto

Explicación del mecanismo de formación de las triples hélices de ARN

Los ARN no codificantes de cadena larga componen gran parte del transcriptoma humano. Estas moléculas de ARN interactúan con la cromatina para regular la expresión génica a través de la formación de hélices triples. A pesar de las numerosas investigaciones «in vitro», siguen sin conocerse los mecanismos subyacentes a la formación de los tríplex. El proyecto triloci-seq, financiado por las Acciones Marie Skłodowska-Curie, trabaja en una técnica de secuenciación de próxima generación para descifrar el código del tríplex «in vivo». El éxito del proyecto depende de la disponibilidad de genomas mamíferos que puedan albergar cientos de miles de posibles sitios objetivo tríplex de alta afinidad y de oligonucleótidos que contengan bases mixtas. Conjuntamente estos recursos deberían permitir que los científicos superen el mayor obstáculo en los estudios de la formación de tríplex, es decir, la necesidad de emparejar específicamente un sitio objetivo con su oligonucleótido formador de tríplex.

Objetivo

Recent technological advances revealed that there are many long non (protein)-coding RNA molecules (lncRNA) transcribed in the genome, many of whom regulate gene expression. However, it remains unclear how lncRNA molecule can interact directly with chromatin to regulate gene expression. One possibility that has been raised is via the formation of triple-helices. The potential for the formation of triple helices exists in any nucleic acid chain via an interaction called Hoogsteen base-pairing. However, unlike the genetic code or even Watson-Creek base-pairing, the triplex code or sequences which can function as triplex target sites and triplex-forming oligos is poorly understood. To decipher the triplex code in vivo, I propose a novel research approach based on next generation sequencing that I call Triloc-seq (in vivo). The feasibility this research plan relies on two crucial resources: first, mammalian genomes which can harbor as many as hundreds of thousands of putative high-affinity triplex target sites (TTSs), and second mixed-base oligo synthesis technology. Together these resources will allow us to over-come the greatest obstacle in triplex study, the need to match specifically a target site with its triplx forming oligo (TFO). To study the triple-helix code, we will transfect mammalian cells with a TFO libraries, and use click chemistry to join the TFO to its target site. We will then use previous TFO-TTS data obtained in vitro and multiple advanced bioinformatic approaches to extract the genomic TTSs. Finally, to prove that triplex interaction can be functional in vivo, we will design several applications that will test this functionality. In all cases we will design a cassette of known TTS target sites and position it upstream of a minimal promoter. The TTS cassette will be targeted by natural lncRNAs containing matching TFO segments, synthetic TFO-VP64 hybrids synthesized in vitro, and fluorescently labelled TFOs to confirm TTS-TFO functionality.