Combinatorial Selections of Structure-Specific Nucleic Acid Binding Ligands – CASTing

Nucleic acids represent strategic molecular targets to understand and combat pathological disorders. The development of highly selective probes and drugs to detect and disturb nucleic acid related processes is then of profound importance to fundamental biology, clinical diagnostics and therapeutics. In the past decade, there has been growing evidence that the formation of high order nucleic acid structures, triplexes and tetraplexes, made of triple or quadruple strands of DNA and/or RNA, profoundly influences cell regulation. Hundreds of thousands of such structured motifs are suspected to form in key regulatory regions of human cells and their occurrences have been compellingly associated with several human diseases such as cancers, viral infections and degenerative disorders.
In this context, we are facing the crucial need to develop new classes of structure-specific nucleic acid binding molecules (“binders”) that could provide reliable insights into the molecular functions and biological significance of triplexes and tetraplexes. While the current “state-of-the-art” structure-specific nucleic acid binders may be able to distinguish duplex DNA and RNA from higher order motifs, they do not discriminate amongst the diverse subclasses of multi-stranded nucleic acid structures. Given the abundance and diversity of nucleic acid polymers in cells, this lack of specificity is a cause for off-targets effects and precludes the unambiguous evaluation of triplex and tetraplex occurrences and functions.
In this context and through an interdisciplinary program, at the crossroads of synthetic chemistry, molecular engineering, combinatorial chemistry and molecular biology, it is my ambition to design, synthesize and exploit smart molecular ligands with unprecedented ability to specifically recognize and affect nucleic acid structures of outstanding biological interest.
To achieve this purpose, I first intend to use synthetic approaches to assemble a representative subset of structured nucleic acid targets (Task 1). To do so, I will implement the most advanced biorthogonal ligation procedures to stabilize biologically relevant triplexes and tetraplexes in their native conformations. These topologically controlled triplexes and tetraplexes will then be used as targets in cutting-edge ligand selection procedures.
I will bridge the gap between rational and combinatorial approaches to identify structure-specific binders from two distinct sets of molecular ligands.
On one hand, we will assemble a large and thoughtfully designed library of over one hundred million small molecules equipped with an optimum chemical diversity to engage in specific binding with all subclasses of multi-stranded nucleic acid motifs (Task 2). This focused library of novel branched molecules, generated by the stepwise association of notorious nucleic acid binding motifs with functionally diverse molecular fragments, will be barcoded to allow for the rapid and cost-efficient identification of highly specific ligands through iterative selection procedures.
On the other hand, we will assemble a library a biostable modified nucleic acid aptamer binding agents uniquely equipped with the optimum chemical diversity to engage in extended discriminative interactions with distinct triplex and tetraplex targets (Task 3). This novel library of base and sugar modified oligonucleotides will be subjected to in vitro selection procedures (SELEX) against the distinct targets assembled in Task 1 to reach the desired specific binding profile.
To successfully identify ligands with unmatched molecular recognition capacities, we will need to address fundamental questions and methodological challenges. The expected breakthrough in nucleic acid recognition is expected to open promising horizons to understand as yet unresolved biological processes, paving the way to the development of novel diagnostic and therapeutic tools.