Fig. 3.

Assessing histidine tag recruiting for the 8XJ105 deoxyribozyme. See the Experimental section for explanation of error bars in all panels. Conditions: 20 nM 3′-³²P-radiolabeled 5′-pppRNA, 0.5 μM 8XJ105 deoxyribozyme, 2 μM 3′-tris(NTA) DNA anchor oligonucleotide, 1–5000 μM peptide, 70 mM HEPES, pH 7.5, 40 mM MgCl₂, 20 mM MnCl₂, 1 mM ZnCl₂, 150 mM NaCl, and CoCl₂, NiCl₂, or Cu(NO₃)₂ as indicated at room temperature. CuCl₂ gave equivalent outcome. (A) Yield at 16 h of peptide-RNA conjugate as a function of concentration of His₆-tagged 24-mer peptide (or untagged 24-mer in one case). All experiments including the controls were performed with 1 mM Zn²⁺; 10 μM Co²⁺, Ni²⁺, or Cu²⁺ was additionally present where indicated. Inset: PAGE data at 16 h for 3′-³²P-radiolabeled RNA and 30 μM His₆-tagged 24-mer peptide with Cu²⁺ (S = substrate, P = product). See Fig. S1 (ESI^†) for data with 5′-tris(NTA) moiety. 24-mer H₆SAGERASAEDMARAAYAA (for non-His₆, replace H₆ with ASAASA); 18-mer H₆SAEDMARAAYAA; 11-mer H₆AAYAA. (B) Optimization of Cu²⁺ concentration, and demonstration that Cu²⁺ alone [without tris(NTA)] is not responsible for the recruiting effect. (C) Determination of peptide K_m values using initial-rate kinetics. See curve fit descriptions and fit details in the Experimental section.