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Structure-based investigation of fluorogenic Pepper aptamer

Nature Chemical Biology volume 17, pages 1289–1295 (2021)Cite this article

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Abstract

Pepper fluorescent RNAs are a recently reported bright, stable and multicolor fluorogenic aptamer tag that enable imaging of diverse RNAs in live cells. To investigate the molecular basis of the superior properties of Pepper, we determined the structures of complexes of Pepper aptamer bound with its cognate HBC or HBC-like fluorophores at high resolution by X-ray crystallography. The Pepper aptamer folds in a monomeric non-G-quadruplex tuning-fork-like architecture composed of a helix and one protruded junction region. The near-planar fluorophore molecule intercalates in the middle of the structure and is sandwiched between one non-G-quadruplex base quadruple and one noncanonical G·U wobble helical base pair. In addition, structure-based mutational analysis is evaluated by in vitro and live-cell fluorogenic detection. Taken together, our research provides a structural basis for demystifying the fluorescence activation mechanism of Pepper aptamer and for further improvement of its future application in RNA visualization.

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Fig. 1: Secondary and tertiary structure of Pepper aptamer in complex with HBC.
Fig. 2: Metals involved in interaction in the tertiary structure of Pepper aptamer in complex with HBC.
Fig. 3: Architecture of the HBC-binding pocket.
Fig. 4: In vitro fluorescence assay of the structure-based mutants of Pepper aptamer.
Fig. 5: The binding-pocket composition of HBC-like fluorophores.

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Data availability

Atomic coordinates and structure factors for Pepper aptamer in complex with HBC and the analogs have been deposited at the Protein Data bank (www.rcsb.org) under accession numbers 7EOH, 7EOK, 7EOL, 7EOM, 7EON, 7EOO and 7EOP. The crystal structures of Ir(NH3)63+-soaked, Mn2+-soaked and Cs+-soaked crystals have been deposited at the Protein Data bank under accession numbers 7EOG, 7EOI and 7EOJ, respectively. Source data are provided with this paper.

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Acknowledgements

We thank the staff members of the Large-scale Protein Preparation System, BL-17B, BL-18U1 and BL-19U1 beamlines at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Laboratory, China for providing technical support and assistance in data collection and analysis. We thank the staff of the BL-17U1 beamline at SSRF for their assistance in X-ray data collection. We thank the core facility of the Life Sciences Institute (LSI), Zhejiang University, for technical assistance. This work was supported by the National Natural Science Foundation of China (32022039, 31870810, 91940302 and 91640104 to A.R., 91857202 and 21937004 to Y.Y.), the National Key Research and Development Program of China (2017YFA050400 and 2019YFA0904800 to Y.Y., 2019YFA0110500 to L.Z.), the outstanding youth fund of Zhejiang Province (LR19C050003 to A.R.), the Shanghai Science and Technology Commission (18JC1411900 to Y.Y.) and the new faculty start-up funds from Zhejiang University (to A.R.), Shanghai Municipal Education Commission-Frontier Research Base of Optogenetic Techniques for Cell Metabolism (2021 Sci & Tech 03-28 to Y.Y. and X.C.) and the Fundamental Research Funds for the Central Universities (to Y.Y. and X.C.).

Author information

Author notes

  1. These authors contributed equally: Kaiyi Huang, Xianjun Chen.

Authors and Affiliations

  1. Life Sciences Institute, Zhejiang University, Hangzhou, China

    Kaiyi Huang, Chunyan Li, Qianqian Song & Aiming Ren

  2. Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China

    Xianjun Chen, Huiwen Li, Linyong Zhu & Yi Yang

  3. CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China

    Xianjun Chen, Huiwen Li & Yi Yang

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Contributions

K.H. undertook all of the crystallization screening and optimization, the diffraction data collection, the structure determination and the in vitro characterization of Pepper aptamer with the assistance of C.L. and Q.S. under the supervision of A.R. X.C. performed the live-cell characterization of Pepper aptamer with the assistance of H.L. under the supervision of Y.Y. L.Z. synthesized HBC and the analogs. The structures were analyzed by A.R. and K.H. The paper was written jointly by A.R. and Y.Y. with input from the remaining authors.

Corresponding authors

Correspondence to Linyong Zhu, Yi Yang or Aiming Ren.

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Competing interests

Y.Y., L.Z. and X.C. are named inventors of patent applications nos. 201910352348X, 201910348701.7, PCT/CN2020/087311, PCT/CN2020/087415. The remaining authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Charles Dann and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The sequence and structure of Pepper aptamer.

a, The sequence and predicted secondary structure of the original selected Pepper aptamer. b, The tertiary structure of Pepper aptamer is shown in ribbon representation with the bases of the residues shown in stick and the bound HBC shown in sphere representation. Stems P1, P2, P3 and the zipped junction J1/2 and J2/1 stacks continuously and form the long helix.

Extended Data Fig. 2 Anomalous difference electron density map for Ir(NH3)63+-soaked, Mn2+-soaked and Cs+-soaked Pepper aptamer crystals.

a, Anomalous electron density map contoured at level 3.0 σ for Ir(NH3)63+ sites (labelled with red arrow) in each asymmetric unit of Pepper aptamer were used to determine the phase problem of the structure. b, Anomalous electron density map contoured at level 3.0 σ for Mn2+ sites (shown as purple balls, labelled with red arrow) in each asymmetric unit of Pepper aptamer. c, Anomalous electron density map contoured at level 3.0 σ for Cs+ sites (shown as cyan balls) in each asymmetric unit of Pepper aptamer. The bound Mg2+ sites are shown as green ball and labelled with red arrow.

Extended Data Fig. 3 Structural details of Pepper aptamer.

a, b, Interaction between junction J3/2 and stem P2. A31 (J3/2) forms one hydrogen bond with the phosphate of G37 (stem P2). N3 of U32 forms one hydrogen bond with O4 of U35 within J3/2. Then, U35 forms two additional hydrogen bonds with the base of G37 and the phosphate of C38 from stem P2. c, A base triple is formed by G34 (J3/2) and the base pair G39-C22 from stem P2. d, C33 (J3/2) forms one hydrogen bond with U40 from the terminal of stem P2 and three additional hydrogen bonds with the Watson-Crick edge of G9 from J1/2, hence a base quadruple G10-U40-C33-G9 is formed in the bottom of J3/2. e, Three consecutive nucleotides G41, U42 and C43 from J2/1 are positioned in the same plane and form hydrogen-bonding interactions with the Watson-Crick edge of U8 from J1/2. f, G44 (J2/1) forms a canonical Watson-Crick base pair with C7 (J1/2). The base of A6 forms extensive interaction with the phosphate of C5, the base of C7 and the sugar of G41. Additionally, the sugar of U42 forms one hydrogen bond with the base of G44. It is notable that A6, G41 and U42 adopt 2’-endo sugar pucker conformation in the structure. g, 2’-OH of A6 pointes upwards and forms hydrogen bond interaction with the base of U8 and G9. The base of A6 forms two hydrogen bonds with the sugar of G41 and the phosphate of C5.

Extended Data Fig. 4 The identified divalent metals and the involved interaction in Pepper structure.

a, The composite omit electron density map (contoured at level 1.0 σ) for four metal cations M1, M2, M3 and M4 (shown as green balls) and the coordinated residues in Pepper aptamer. b, M5 located in the major groove of stem P1 and forms indirect coordination with the phosphates of G45, G46, G47, G48 and the base of G47, G2 from stem P1.

Extended Data Fig. 5 The metal ions dependence assay of Pepper aptamer.

a-d, The fluorescence activation assay of HBC by Pepper aptamer in the presence of Mg2+ (a), Mn2+ (b), Ca2+ (c) and Ba2+ (d). 50 nM refolded RNA in the reaction buffer containing 40 mM HEPES, pH 7.4, 125 mM KCl supplemented with individual divalent metal ions at different concentration ranging from 0 mM to 10 mM was titrated with increasing concentrations of HBC ranging from 0 μM to 2 μM. e, The activated fluorescence of HBC by Pepper aptamer in the presence of K+ at different concentration from 0 mM to 50 mM. The titration of 50 nM refolded RNA with HBC (concentrations ranging from 0 μM to 2 μM) in the reaction buffer containing 40 mM HEPES, pH 7.4, 5 mM MgCl2 supplemented with 0 mM to 50 mM KCl was carried out. Three independent experiments were carried out with similar results. Data represent the mean ± s.d. from three replicates.

Source data

Extended Data Fig. 6 The composition of HBC binding pocket in Pepper tertiary structure.

a, b, Two rotated views of the four-sided binding box shown in Fig. 3c that capsulate ligand HBC in the tertiary structure. c, d, The binding box of HBC is further wrapped by the base triples G39-C11-G34 and A6-C7-G44 between stem P2 and the zipped junction J1/2 and J2/1. e, f, The composite omit electron density map (contoured at 1.0 σ level) of the residues involved in the formation of HBC-binding pocket (e) and the bound HBC (f) are shown respectively.

Extended Data Fig. 7 Effect with deoxynucleotide mutations in Pepper aptamer.

a, The activated fluorescence of HBC by Pepper mutants A6dA, G41dG and U42dU from three independently-repeated experiments are normalized for comparison with the wild-type (WT) Pepper aptamer. b, The titration of Pepper mutants A6dA, G41dG and U42dU with HBC were carried out in the buffer containing 40 mM HEPES, pH 7.4, 125 mM KCl and 5 mM MgCl2 at room temperature. All the titrations are performed three times independently. Data represent the mean ± s.d. from three replicates.

Source data

Extended Data Fig. 8 The secondary structure of Pepper aptamer and selected mutants M18-24.

a, The secondary structure of Pepper aptamer used in crystallization. b-d, The secondary structure of Pepper aptamer mutants M18 (b), M19 (c) and M20 (d) with truncated stem P1. e, f, The secondary structure of Pepper aptamer mutants M21 (e) and M22 (f) with truncated stem P3. g, The secondary structure of Pepper aptamer mutant M23, in which the bulge junction J3/2 is deleted. h, The secondary structure of Pepper aptamer mutant M24, in which the bulge junction J3/2 and stem P3 are deleted.

Extended Data Fig. 9 In vitro and in vivo fluorescence assay of selected mutants for M18-M24.

a, Structure-based mutants of Pepper aptamer focusing on the truncation of Pepper aptamer. b, The in vitro fluorescence activation of HBC by Pepper mutants are normalized for comparison with the wild-type (WT) Pepper aptamer. All the tests are performed three times independently. Data represent the mean ± s.d. from three replicates. c, FACS analysis of Pepper fluorescence in mammalian cells. HEK293T cells transfected with plasmid expressing Pepper or Pepper variant were incubated with 1 μM HBC and analyzed using a flow cytometer. HEK293T cells transfected with mock plasmid and labeled with 1 μM HBC were used as the controls. Gate (green) was placed based on the control cells to determine the population fraction that exhibited higher fluorescence than the background.

Source data

Extended Data Fig. 10 The binding pockets composition of HBC-like fluorophores.

The chemical structures, the spectral properties and the composition of the binding pockets of HBC514 (a), HBC508 (b), HBC497 (c), HBC620 (d), HBC525 (e) and HBC485 (f) in rotated view as shown in Fig. 5. All the modification sites of HBC-like fluorophores are labelled in red in the chemical structure and shown with transparent ball in the binding pocket composition. The excitation (Ex.) and emission (Em.) wavelengths, and the binding dissociation constant of each HBC-like fluorophore were shown with the compound as well.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Data 1

wwPDB validation report for 7EOG.

Supplementary Data 2

wwPDB validation report for 7EOH.

Supplementary Data 3

wwPDB validation report for 7EOI.

Supplementary Data 4

wwPDB validation report for 7EOJ.

Supplementary Data 5

wwPDB validation report for 7EOK.

Supplementary Data 6

wwPDB validation report for 7EOL.

Supplementary Data 7

wwPDB validation report for 7EOM.

Supplementary Data 8

wwPDB validation report for 7EON.

Supplementary Data 9

wwPDB validation report for 7EOO.

Supplementary Data 10

wwPDB validation report for 7EOP.

Source data

Source Data Fig. 4

Normalized fluorescence intensity, source data.

Source Data Extended Data Fig. 5

Normalized fluorescence intensity, source data.

Source Data Extended Data Fig. 7

Normalized fluorescence intensity, source data.

Source Data Extended Data Fig. 9

Normalized fluorescence intensity, source data.

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Huang, K., Chen, X., Li, C. et al. Structure-based investigation of fluorogenic Pepper aptamer. Nat Chem Biol 17, 1289–1295 (2021). https://doi.org/10.1038/s41589-021-00884-6

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