Enzymatic synthesis of reactive RNA probes containing squaramate-linked cytidine or adenosine for bioconjugations and cross-linking with lysine-containing peptides and proteins

Figures

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  Fig. 1. Overview of related reactive DNA and RNA probes. A Previous related works on reactive DNA and RNA probes and (B) conceptualization of this study: polymerase synthesis of squaramate-containing reactive RNA probes and their cross-linking with Lys-containing peptides or proteins.

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  Fig. 2. Synthesis of modified NTPs and RNA probes. A Synthesis of CESQTP and AESQTP. B IVT reactions with CESQTP or AESQTP and T7 RNAP. C PAGE analysis of IVT reactions. (+) positive controls with natural NTPs, (−) negative controls in the absence of CTP or ATP, (1CESQ, 3CESQ) IVT reactions with CESQTP, UTP, GTP, and ATP, (1AESQ, 3AESQ) IVT reactions with AESQTP, UTP, GTP, and CTP. D PEX reactions with CESQTP or AESQTP and TGK polymerase. E PAGE analysis of PEX reactions. (P) primer, (+) positive control with natural NTPs, (C−) negative control in the absence of CTP, (A−) negative control in the absence of ATP, (CESQ) PEX reaction with CESQTP, UTP, GTP and ATP, (AESQ) PEX reaction with AESQTP, UTP, GTP and CTP. For the uncropped gels see Supplementary Fig. 1.

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  Fig. 3. Reactivity and specificity of modified RNA probes. Cross-linking reaction scheme of ESQ-modified RNA with (A) amine-linked Cy5 fluorophore, (C) Lys-containing peptide, and (E) crossreactivity in comparison to CA-modified RNA with Cys-containing GSH. B) Fluorescence measurement of cross-linked 35_RNA_1CESQ_Cy5 (red line) and non-cross-linked natural RNA (black line). D) PAGE analysis of cross-linking reactions of 35_RNA_1CESQ with AcKAA peptide. (lane 1) 35_RNA_1CESQ; (lane 2) cross-linking reaction in borate or (lane 3) carbonate buffer. F) PAGE analysis of crosslinking reactions of 35_RNA_1AESQ and 35_RNA_1ACA with AcKAA and GSH. (lane 2) 35_RNA_1AESQ, (lane 3) 35_RNA_1AESQ with AcKAA, (lane 4) 35_RNA_1AESQ with GSH, (lane 5) 35_RNA_1ACA; (lane 6) 35_RNA_1ACA with AcKAA, (lane 7) 35_RNA_1ACA with GSH. For the uncropped gels see Supplementary Fig. 1.

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  Fig. 4. Reactivity of modified RNA probe with recombinant proteins. A Cross-linking of ESQ-modified RNA (36_vRNA_1gCESQ) with Lys-containing protein. B 10% SDS-PAGE analysis of cross-linking experiments with natural or ESQ-modified RNA and various recombinant proteins (2–20 equiv. of protein; for the uncropped gels see Supplementary Fig. 1).

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  Fig. 5. Summary of RdRp assay. A General scheme of RdRp polymerase assay. Hairpin RNA substrate with template-product scaffold. B Gel-based JEV RdRp assay analyzed by western blot and SDS-PAGE (lane C) positive control – reaction with natural NTPs; (lane C*) reaction with CESQTP, GTP, ATP, UTP. For the uncropped gels see Supplementary Fig. 1. C Proteinase K or Trypsin digestion of the RNA-protein conjugate; (lane 1) template; (lane 2) negative control – reaction without CTP; (lanes 3 and 5) positive control – reaction with natural NTPs; (lanes 4 and 6) reaction with CESQTP, GTP, ATP, UTP. D RNA digestion for nano LC-MS/MS analysis and spectrum of 46_RNA_HPC4_JEVNS5 with identification of K269 (m/z acquired: 586.26 Da; Supplementary Fig. S45).