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Triloci-seq - Dechipering the triple helix code

Objective

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.

Field of science

  • /natural sciences/biological sciences/genetics and heredity/genome
  • /natural sciences/biological sciences/biochemistry/biomolecules/nucleic acid

Call for proposal

H2020-MSCA-IF-2018
See other projects for this call

Funding Scheme

MSCA-IF-GF - Global Fellowships

Coordinator

TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD
Address
The Senate Building Technion City 1
32000 Haifa
Israel
Activity type
Private for-profit entities (excluding Higher or Secondary Education Establishments)
EU contribution
€ 181 365,12

Partners (1)

MASSACHUSETTS INSTITUTE OF TECHNOLOGY
United States
Address
Massachusetts Avenue 77
02139 Cambridge
Activity type
Higher or Secondary Education Establishments