Advances in DNA binding by threading polyintercalation

Chemistry

Although molecules that bind DNA have the potential to modify gene expression, the reality of targeting DNA in a sequence-specific manner is still a problematic but worthwhile goal. The Iverson lab has been exploring DNA recognition through a motif known as threading polyintercalation based on connecting intercalating naphthalene diimide (NDI) units, which are molecules that insert themselves between DNA base pairs, together with peptide linkers. These polyintercalators interact with both DNA grooves by “threading” or winding through the DNA, like a snake might climb a ladder. Initially, two different bisintercalator modules with altered sequence specificities and different groove binding topologies were discovered and used to inspire the design of a hybrid NDI tetraintercalator. Surprisingly enough, this tetraintercalator bound sequence-specifically with a dissociation half-life of 16 days to its preferred 14 bp site, a record at the time it was reported for a synthetic DNA-binding molecule. The work reported here expands on the capabilities of this modular threading polyintercalation motif. Chapter 2 describes the ability of a new hybrid NDI tetraintercalator, where the bisintercalator modules are connected together in a different way compared to the previously studied tetraintercalator, to subtly discriminate between similar binding sites. Chapter 3 offers a structural understanding, through NMR analysis, for the sequence recognition abilities of this new tetraintercalator. Chapter 4 analyzes the binding abilities of an un-optimized NDI octaintercalator and proposes how to approach the second-generation design of longer polyintercalators. Chapter 5 describes the optimization of the originally designed NDI tetraintercalator by serially lengthening one of the linkers to produce a tetraintercalator with a 57 day dissociation half-life from its 14 bp sequence, a new record for a synthetic DNA-binding molecule. Using the optimized linker in the context of an NDI hexaintercalator allows for binding to a 22 bp designed site, a record for a synthetic non-nucleic acid molecule. Chapter 6 recounts a focused library screening to search for bisintercalators with new sequence specificities. These efforts have laid the groundwork to progress toward studies aimed at understanding how these molecules might function to prevent transcription in a sequence-dependent manner in vivo.