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G-quadruplexes (G4s) are four-stranded DNA secondary structures that occur in the human genome and play key roles in transcription, replication, and genome stability. G4-specific molecular probes are of vital importance to elucidate the structure and function of G4s. The scFv antibody BG4 has been a widely used G4 probe but has various limitations, including relatively poor in vitro expression and the inability to be expressed intracellularly to interrogate G4s in live cells. To address these considerations, we describe herein the development of SG4, a camelid heavy-chain-only derived nanobody that was selected against the human Myc DNA G4 structure. SG4 exhibits low nanomolar affinity for a wide range of folded G4 structures in vitro. We employed AlphaFold combined with molecular dynamics simulations to construct a molecular model for the G4-nanobody interaction. The structural model accurately explains the role of key amino acids and Kd measurements of SG4 mutants, including arginine-to-alanine point mutations that dramatically diminish G4 binding affinity. Importantly, predicted amino acid-G4 interactions were subsequently confirmed experimentally by biophysical measurements. We demonstrate that the nanobody can be expressed intracellularly and used to image endogenous G4 structures in live cells. We also use the SG4 protein to positionally map G4s in situ and also on fixed chromatin. SG4 is a valuable, new tool for G4 detection and mapping in cells., Herchel Smith funds

The process of displaying functional peptides by 'grafting' them onto loops of a stable protein scaffold can be used to impart binding affinity for a target, but it can be difficult to predict the affinity of the grafted peptide and the effect of grafting on scaffold stability. In this study, we show that a series of peptides that bind to the E3 ubiquitin ligase Keap1 can be grafted into the inter-repeat loop of a consensus-designed tetratricopeptide repeat (CTPR) protein resulting in proteins with high stability. We found that these CTPR-grafted peptides had similar affinities to their free peptide counterparts and achieved a low nanomolar range. This result is likely due to a good structural match between the inter-repeat loop of the CTPR and the Keap1-binding peptide. The grafting process led to the discovery of a new Keap1-binding peptide, Ac-LDPETGELL-NH2, with low nanomolar affinity for Keap1, highlighting the potential of the repeat-protein class for application in peptide display., CRUK Pioneer Award (C17838/A22676) CRUK Biotherapeutic Drug Discovery Project Award (C17838/A27225). BBSRC Doctoral Training Scholarship (BB/J014540/1

c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy. Here we reassess the challenges of directly targeting c-Myc, evaluate lessons learned from current inhibitors, and explore how future strategies such as miniaturisation of Omomyc and targeting E-box binding could facilitate translation of c-Myc inhibitors into the clinic.