Since the conserved groove of NOT9 has been suggested to be the binding site for both CBMs and nucleic acid, we wanted to investigate whether the NIP-2-Ac peptide could compete with RNA binding. To this end, we first investigated the binding efficiency of the previously described model RNA referred to as A20 which has the sequence UCUAAAU followed by 20 adenosine residues and a 5'-FAM label. At low salt concentrations (10 mM NaCl) A20 exhibited considerable binding (KD = 525 ± 157 nM, supplemental Fig. ) as determined by FP while the shorter A0 RNA lacking the poly(A) tail still bound but with a 3.4 fold lower affinity (KD = 1837 ± 455 nM). This seemed to indicate that RNA-binding to NOT9 is not driven by the poly(A) tail itself but rather by length of the RNA and therefore mainly by electrostatic interactions. To further confirm this, we evaluated the affinity of two RNA sequences where the poly(A) tail was exchanged for pyrimidines with either a poly(C) or poly(U) stretch (C20 and U20 respectively). For both RNAs a binding affinity similar to that of A20 was observed (C20: KD = 798 ± 212 nM; U20: KD = 569 ± 86 nM, supplemental Fig. ). The equal potency for RNAs of equal length but different identity suggests that RNA-binding by NOT9 is non-specific. Indeed, a large positively charged patch on NOT9 is thought to mediate RNA-binding which would suggest that binding occurs via the phosphate diester backbone rather than through specific interaction with RNA bases. The substantially higher affinity of NIP-2-F suggested it should be able to compete with RNA-binding and a competitive FP experiment indeed showed that NIP-2-Ac efficiently displaces the A20 RNA with an IC50 value of 333 ± 34 nM. NIP-WT-Ac was slightly more potent (IC50 = 176 ± 29 nM) while NIP-MUT-Ac (IC50 = 5682 ± 444 nM) was a lot weaker as expected (Fig. ). Nonetheless, the negative control peptide was not fully inactive potentially due to non-specific interaction between the positive patch on NOT9 and the strongly negatively charged peptide. This is in line with previous results using electrophoretic mobility shift assays where the negative control peptide also still moderately inhibited RNA binding. 24 To further validate the interaction between NIP-2-Ac and NOT9, we sought to determine the co-crystal structure. We chose the NOT9-NOT1 complex for co-crystallization as NOT9 alone tends to dimerize according to an earlier report. We obtained crystals and solved the structure of the NIP-2-Ac peptide bound to NOT9 using data until 2.64 Å (Fig. ). The asymmetric unit contained two structurally similar NOT9-NOT1-NIP-2-Ac complexes with an RMSD of 0.533 Å over the C α -atoms. In both complexes, the mode of interaction of NIP-2-Ac with the concave surface of the NOT9 is similar and no contacts between NIP-2-Ac and NOT1 were observed. Both the backbone fold of NIP-2-Ac as well as the side chain orientations closely match the reported wild type α-helical Bam CBM domain bound to the NOT9-NOT1 complex (PDB 5ONA) and the same hydrophobic core is responsible for binding with the NOT9 surface (Fig. ). The crystal structure also allowed us to detect the electron density of the hydrocarbon staple which sits at a site orthogonal to the NIP-2-Ac -NOT9 binding interface as the design intended (see supplemental Fig. ). Apart from hydrophobic interactions, the R130 guanidine from NOT9 contributes to binding via a cation-pi interaction with NIP-2-Ac F21 and a potential salt-bridge with D18 (only observed in one dimer in the asymmetric unit, Fig. ). Additionally, the side-chain amides of NIP-2-Ac N20 and NOT9 N88 form a hydrogen bond, which is also observed in the Bam-NOT9 interaction. In summary, the preorganization of the helical fold by hydrocarbon stapling in NIP-2-Ac correctly mimics the backbone fold of the CBM domain of the Bam protein.