β-Alanine and N-terminal cationic substituents affect polyamide-DNA binding

Abstract

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as β-alanine (β) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of β and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that β inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a β replacement between two imidazole heterocycles (ImβIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of βs and cationic substituents.

Fig. 1

A)Systematically designed polyamides that are categorized into two modified groups: β-alanine inserts (red) and cationic substitutions (blue). The illustration above KA2035 is the simplified representative of KA2035. The open and closed circles stand for pyrrole and imidazole, respectively. The diamond represents β-alanine. The numbering starting from N-terminus to C-terminus applies to all PAs. B) The biotinylated cognate binding sequence of the PAs: λB DNA. The predetermined PA binding site is highlighted in the green frame.

Fig. 2

Representative SPR sensorgrams of PAs binding to λB DNA. From left to right, there are strong (K_D = 0.67 nM), intermediate (K_D = 1.62 nM), and weak (K_D = 4.1 nM), binders. The colored lines are experimental sensorgrams. The black overlays are 1:1 global kinetic fits.

Fig. 3

A) Direct comparison of equilibrium affinities of all PAs binding to λB sequence. The grouped red and blue columns represent molecules that have the same β modifications but different N-termini. B) Binding affinities of all PAs to λB, SC1 and GAGA mutant sequences. The binding of PAs in β-alanine inserts group to SC1 sequence is comparable to that of λB and GAGA mutant sequences. While extremely weak, some even undetectable binding was observed for PAs in the cationic substitutions group with SC1 sequence. Note that KA2035 is included into β-inserts group for ease of comparison.

Fig. 4

CD spectra of PA titration with short λB sequence (5’-GGAAGTGAACCTCTGTTCACTTCC-3’).