Synergistic DNA- and Protein-Based Recognition Promote an RNA-Templated Bio-orthogonal Reaction

Abstract

Biomolecular assemblies composed of proteins and oligonucleotides play a central role in biological processes. While in nature, oligonucleotides and proteins usually assemble via non-covalent interactions, synthetic conjugates have been developed which covalently link both modalities. The resulting peptide-oligonucleotide conjugates have facilitated novel biological applications as well as the design of functional supramolecular systems and materials. However, despite the importance of concerted protein/oligonucleotide recognition in nature, conjugation approaches have barely utilized the synergistic recognition abilities of such complexes. Herein, the structure-based design of peptide-DNA conjugates that bind RNA through Watson-Crick base pairing combined with peptide-mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics have been designed to adjacently bind a particular RNA sequence. In the resulting ternary complex, their peptide elements are located in proximity, a feature that was used to enable an RNA-templated click reaction. The introduced structure-based design approach opens the door to novel functional biomolecular assemblies.

Keywords: RNA recognition; bio-conjugation; peptides; self-assembly; strain-promoted click reaction.

Figure 1

(a) Model of two identical TAV2b fragments (aa 21–53, sTAV2b, orange) bound to duplex RNA (grey). Model was derived from crystal structure PDB ID: 2ZI0. (b) Oligonucleotide sequences used to test duplex binding (RNA: gray, DNA: blue). (c) Representative ITC plots of complex formation between sTAV2b (c=108 μM) and RNA^α/DNA^β (c(duplex)=6 μM). Measurements were performed in triplicate, errors=1σ; for full data see Supporting Information Table S4 and Figure S4. Buffer: Phosphate‐buffered saline (pH 7.4) (d) CD spectra of sTAV2b (c=4 μM; orange line), RNA^α/DNA^β (c(duplex)=2  μM; blue line), spectra of RNA^α/DNA^β (c(duplex)=2 μM) with sTAV2b (c=4 μM) (black line) and the sum of the two individual spectra (dotted black line). Buffer: 10 mM sodium phosphate (pH 7.4), 100 mM NaCl.

Figure 2

(a) Schematic overview of the design of conjugate type A and B: sTAV2b (orange) bound to duplex RNA/DNA (grey/blue) serves as starting point for the design of peptide‐DNA conjugates (1‐A12 and 2‐B12) targeting single stranded RNA^α (grey). Chemical structure of linkages between peptide and DNA are shown. (b) EMSA of RNA^α in the presence and absence of DNA A12, a mixture of DNA and peptide (A12+1) and conjugate 1‐A12. Experiments employed 15 % native polyacrylamide gel electrophoresis (PAGE) (c(RNA)=3 μM, c(ligand)=4.5 μM. Running buffer: 1x TAE, stain: SYBR gold (full gel in Supporting Information Figure S8). (c) K _d‐values determined by ITC for RNA^α‐ligand interactions (triplicate measurements, errors=1σ; for full data see Supporting Information Table S4 and Figures S9‐11). (d) Representative ITC of 1‐A12 with the RNA^α (for full data see Supporting Information Figure S11). (e) EMSA of RNA^α in the presence and absence of DNA B12, a mixture of DNA and peptide (B12 + 2) and conjugate 2‐B12 (for details see caption Figure 2b, full gel in Supporting Information Figure S12). (f) K _d‐values determined by ITC for RNA^α/ligand interactions (triplicate measurements, errors=1σ; for full data see Supporting Information Table S4 and Figures S13–15).(g) Representative ITC of 2‐B12 with the RNA^α (for all data see Supporting Information Figure S15).

Figure 3

(a) Melting temperature profiles of RNA^α in the presence of A12, an equimolar mixture of A12+1, and 1‐A12 respectively. (b) Melting temperature profiles of RNA^α in the presence of B12, an equimolar mixture of B12+2, and 2‐B12 respectively (λ=267 nm, c(RNA^α)=2 μM, c(binding partners)=2 μM. Buffer: 10 mM sodium phosphate, 100 mM NaCl (pH 7.4). (c) Sequences of DNA‐truncated A‐family conjugates. Table of T _m‐values of conjugates with RNA^α (for melting curves see Supporting Information Figure S18). (d) Sequences of DNA‐truncated B‐ family conjugates. Table of T _m‐values of conjugates with RNA^α (for melting curves see Supporting Information Figure S18). (e) EMSA of RNA^α in the presence of different conjugates (Supporting Information Figure S19) including co‐incubation of A‐ and B‐series members with DNA sequences of equal lengths. Experiments employed 15 % native PAGE (c(RNA)=3 μM, c(conjugate)=4.5 μM. Running buffer: TAE, stain: SYBR gold. Cartoon representations of the proposed RNA/conjugate complexes corresponding to band species are presented on the right hand side.

Figure 4

(a) Schematic overview of RNA^α‐templated reaction. Azide‐modified conjugates 3 and 4 bind RNA^α to form a ternary complex which reacts with a bis‐alkyne crosslinker to yield a complex between ligated conjugate 3*4 and RNA^α. (b) Structure of heterodimeric product 3*4 including the chemical structure of the reacted crosslink. (c) HPLC traces of unreacted 3 and 4 (t=0) and of reactions between 3, 4 and 5 after 6 h in the presence of RNA^α (red line), in the absence of RNA (no RNA), and in the presence of RNA^β (gray). (d) Electrospray ionization (ESI) mass spectrum of heterodimeric product 3*4 (full characterization see Supporting Information Figure S23). (e) Time‐dependent yields of 3*4 as determined by HPLC including initial rates (v _r). Conjugates (c=5 μM) were incubated for 18 h at room temperature in the presence or absence of an RNA template (c=5 μM) and bis‐alkyne crosslinker 5 (c=5.75 μM). Reaction buffer: 100 mM NH₄HCO₃, 20 % acetonitrile, 1 % DMSO (pH=8.0). Templated reactions were performed in triplicate, errors=1σ; for full data see Supporting Information Table S6 and Figure S27.