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Intricate 3D architecture of a DNA mimic of GFP

Nature volume 618, pages 1078–1084 (2023)Cite this article

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Abstract

Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures1,2,3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates4 conditional fluorophores derived from GFP. To extend previous structural studies5,6 of fluorogenic RNAs, GFP and other fluorescent proteins7 to DNA, we characterize Lettuce–fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding—including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA—and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.

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Fig. 1: Overall structure of the Lettuce fluorescence turn-on DNA aptamer.
Fig. 2: The Lettuce fluorophore-binding site.
Fig. 3: The Lettuce core and its structure-guided minimization.
Fig. 4: Detection of R-loop formation by Lettuce.
Fig. 5: Comparison to RNA and protein counterparts.

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Data availability

Atomic coordinates and structure factor amplitudes have been deposited into the Protein Data Bank (PDB) database under accession codes 8FHV (Lettuce–Tl–DFHBI-1T), 8FHX (Lettuce–DFHBI-1T), 8FHZ (Lettuce–DFHO), 8FI0 (Lettuce–DFAME), 8FI1 (Lettuce C20G–DFHO), 8FI2 (Lettuce C20T–DFHBI-1T), 8FI7 (Lettuce C20T–DFHO) and 8FI8 (Lettuce C20T–DFAME). Cryo-EM data have been deposited into the Electron Microscopy Data Bank (EMDB) database under accession code EMD-29329. The following data used in this study are available at the PDB database under accession codes 1EMA (GFP), 4TS0 (Spinach RNA aptamer), 7OAX (Chili RNA aptamer), 1AW4 (AMP-binding DNA aptamer), 1DB6 (argininamide DNA aptamer), 6J2W (OBA3 DNA aptamer), 7W9N (OBA36 DNA aptamer), 5CKK (RNA-ligating deoxyribozyme 9DB1), 5XM8 (RNA-cleaving deoxyribozyme Dz36) and 5OB3 (iSpinach RNA aptamer). For uncropped gels, see Supplementary Fig. 1. Please refer to Supplementary Table 1 for accession codes and references used in the RNA and DNA compactness study. Source data are provided with this paper.

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Acknowledgements

We thank the staff of beamlines 5.0.1 and 5.0.2 of the Advanced Light Source, Lawrence Berkeley National Laboratory (ALS), and 24-ID-C and 24-ID-E of the Advanced Photon Source, Argonne National Laboratory (APS) for crystallographic data collection; G. Piszczeck and D. Wu of the Biophysics Core of the National Heart, Lung and Blood Institute (NHLBI) for fluorescence and CD; H. Wang and U. Baxa of the NIH Multi-Institute Cryo-EM Facility (MICEF) for cryo-EM data collection assistance; and C. Bou-Nader, N. Demeshkina, A. Elghondakly, C. Jones and R. Trachman for discussions. This research used resources of the APS, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (NIH P30 GM124165). The Pilatus 6M detector on the 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). L.F.M.P. and M.T.B. are Lenfant Postdoctoral Fellows of the NHLBI. This work was supported in part by NIH awards R35NS111631 (to S.R.J.) and T32GM007739 (to J.D.M.) and by the intramural programme of the NHLBI, NIH.

Author information

Author notes

  1. Xing Li

    Present address: Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China

Authors and Affiliations

  1. Laboratory of Nucleic Acids, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Luiz F. M. Passalacqua, Michael T. Banco & Adrian R. Ferré-D’Amaré

  2. Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, NY, USA

    Jared D. Moon, Xing Li & Samie R. Jaffrey

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Contributions

S.R.J. and A.R.F.-D. initiated the project. L.F.M.P. performed fluorescence, crystallographic, CD and cryo-EM experiments and data analyses. M.T.B. performed fluorescence and cryo-EM experiments and data analyses. J.D.M. isolated and characterized the Lettuce sequence. X.L. synthesized fluorophores. A.R.F.-D. and L.F.M.P. wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Adrian R. Ferré-D’Amaré.

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Competing interests

S.R.J. is the co-founder and has equity in Chimerna Therapeutics and Lucerna Technologies. Lucerna has licensed technology related to Spinach and other RNA–fluorophore complexes. All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Wall-eyed stereoviews of Lettuce-DFHBI-1T complex, composite simulated annealing-omit electron density maps for Lettuce-fluorophore complexes, and solution characterization of Lettuce.

Wall-eyed stereoviews of Figs. 1c and d are shown on a, and b, respectively. Composite simulated annealing-omit 2|Fo| - |Fc| electron density map for the c, Lettuce-DFHBI-1T, d, Lettuce-DFHO, e, Lettuce-DFAME, f, Lettuce(C20G)-DFHO, g, Lettuce (C20T)-DFHBI-1T, h, Lettuce (C20T)–DFHO, i, Lettuce (C20T)–DFAME complexes contoured at 1.0 σ (gray mesh), superimposed on the respective final refined models. j, Graphical G-quartet schematics (ref. 17) for Lettuce. Each row represents the nucleotides of a quartet tier, and columns indicate nucleotide stacks. Upper-case, lower-case, bold, and Italic letters denote anti, syn, 2′-endo and 3′-endo nucleotides, respectively; upside-down letters denote strand polarity inversion with respect to the 5′-most nucleotide in the scheme. Lines connecting nucleotides are loops and bulges, with the number of nucleotides indicated within a circle. k, Circular dichroism spectra of Lettuce–DFHBI1T (green) and unliganded Lettuce (gray) recorded at 21 °C. l, Size-exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis of unliganded Lettuce. Dashed green line is absorbance at 280 nm for unliganded Lettuce, and red line is absorbance at 280 nm for tRNALys control. Colored dots correspond to calculated molar mass (red is tRNALys control, and green is unliganded Lettuce). Exclusion limit (V0) for this column is at 4.5 mL (not shown).

Source Data

Extended Data Fig. 2 Single-particle cryo-EM analysis of the unliganded Lettuce aptamer.

a, Image processing workflow for unliganded Lettuce. Processing steps denoted in red and black represent programs used in either cryoSPARC or RELION, respectively. b, Representative of a motion-corrected micrograph from dataset of 3769 dose-weighted micrographs. c, Representative 2D class averages of unliganded Lettuce from the final 2D classification. d, Global resolution assessment by Fourier shell correlation curve with the 0.143 gold standard threshold. e, Distribution of orientations over azimuth and elevation angles for particles included in the calculation of the final map.

Extended Data Fig. 3 Lettuce–DFHBI-1T in complex with thallium (I), overall structures of Lettuce in complex with DFHO and DFAME, ball-and-stick representation of the tiers of Lettuce, and wall-eyed stereoviews of the binding site.

a, Cartoon representation of the Lettuce–DFHBI-1T Tl+ complex. Arrows denote 5′ to 3′ chain direction, brown and green spheres represent Tl+ and Mg2+, respectively. The bound DFHBI-1T molecule is shown in ball-and-stick representation with translucent spheres. DNA color as in Fig. 1c. b, Orthogonal view of a. c, Density-modified SAD electron density map for the Lettuce–DFHBI-1T Tl+ complex contoured at 1.0 σ (blue mesh), superimposed on the final refined model. d, Emission spectra of Lettuce–DFHBI-1T in the presence of K+ (solid green line) or Tl+ (dashed green line). e, Cartoon representation of the Lettuce–DFHO complex. Arrows indicate 5′ to 3′ chain direction, purple and green spheres represent K+ and Mg2+, respectively. The bound DFHO molecule is shown in ball-and-stick representation with translucent spheres. DNA color as in Fig. 1c. f, Orthogonal view of e. g, Cartoon representation of the Lettuce–DFAME complex. Arrows indicate 5′ to 3′ chain direction, purple and green spheres represent K+ and Mg2+, respectively. The bound DFAME molecule is shown in ball-and-stick representation with translucent spheres. DNA color as in Fig. 1c. h, Orthogonal view of g. i, Ball-and-stick representation of the nine tiers (numbered 1-9 in bold numerals) in the Lettuce core. Some background or interaction nucleotides of different tiers are shown for clarity. Gray and orange dashed lines denote hydrogen bonds and metal coordination, respectively. Secondary structure representation corresponds to that in Fig. 1e. Wall-eyed stereoviews of Figs. 2a–d and are shown on j, k, l, and m, respectively.

Source Data

Extended Data Fig. 4 Fluorescence titrations, overall structures and spectra of Lettuce C20 specificity mutants complexed with different fluorophores, and wall-eyed stereoviews of Lettuce C20 specificity mutants binding sites.

Fluorescence of DFHBI-1T (green), DFHO (yellow), and DFAME (red) titrated with a, Lettuce, b, Lettuce C20G mutant, and c, Lettuce C20T mutant (n = 3 technical replicates). d, Calculated Kd values for a, b, c (mean ± s.d., n = 3 technical replicates). e, Cartoon representation of the C20G Lettuce–DFHO complex. Arrows indicate 5′-to-3′ chain direction, purple and green spheres represent K+ and Mg2+, respectively. Asterisk marks the mutated residue. The bound DFHO molecule is shown in ball-and-stick representation with translucent spheres. DNA color as in Fig. 1c. f, Orthogonal view of e. g, Cartoon representation of the C20T Lettuce–DFHO, i, –DFHBI1T, and k, –DFAME complexes. Arrows indicate 5′ to 3′ chain direction, purple spheres represent K+, and green spheres represent Mg2+. The bound fluorophore molecules are shown in ball-and-stick representation with translucent spheres. Asterisk marks the mutated residue. DNA color as in Fig. 1c. h, Orthogonal view of g. j, Orthogonal view of i. l, Orthogonal view of k. m, Excitation and emission spectra of Lettuce and Lettuce C20T mutant in the presence of DFHBI-1T. n, Excitation and emission spectra of Lettuce, Lettuce C20G mutant, and Lettuce C20T mutant in the presence of DFHO. o, Excitation and emission spectra of Lettuce and Lettuce C20T mutant in the presence of DFAME. Wall-eyed stereoviews of Figs. 2g–j and are shown on p, q, r, and s, respectively.

Source Data

Extended Data Fig. 5 Wall-eyed stereoviews of the Lettuce core, divalent cation dependence of Lettuce fluorescence, and Bibb Lettuce characterization.

Wall-eyed stereoviews of Figs. 3a–d are shown on a, b, c, and d, respectively. e, Fluorescence of Lettuce-DFHBI-1T as a function of Mg2+ concentration (in the presence of 150 mM K+). The half-maximal activity (K1/2) is at 1.41 ± 0.14 mM Mg2+ (mean ± s.d.; n = 3 technical replicates). f, Fluorescence activation of Lettuce–DFHBI-1T in 150 mM K+ alone, or supplemented with 10 mM Mg2+, Ca2+, or Mn2+ (mean ± s.d., n = 3 technical replicates). * denotes P= 0.0002 (two-sided t-test). No significancy (P= 0.251) between K+ + Mg2+ and K+ + Mn2+ (two-sided t-test). g, Electrostatic potential of Lettuce mapped on its molecular surface. Color ramp from 0 to +20 kBT (white to red). h, Green sphere shows Mg2+ ion bound in the P1.1 loop. i, Molecular surface of Lettuce (same color scheme as g) showing the P2.1 loop at its center. j, Green sphere shows Mg2+ ion bound in the P2.1 loop. k, Circular dichroism spectra of Bibb Lettuce–DFHBI1T (green) and unliganded Bibb Lettuce (gray) recorded at 21 °C. l, Fluorescence of DFHBI-1T (green), DFHO (yellow), and DFAME (red) titrated with Bibb Lettuce. Calculated Kd values are shown (mean ± s.d., n = 3 technical replicates). m, Fluorescence of Bibb Lettuce-DFHBI-1T as a function of Mg2+ concentration (in the presence of 150 mM K+). The half-maximal activity (K1/2) is at 0.52 ± 0.06 mM Mg2+ (mean ± s.d.; n = 3 technical replicates). n, Fluorescence activation of Lettuce–DFHBI-1T in 150 mM K+ alone, or supplemented with 10 mM Mg2+, Ca2+, or Mn2+ (mean ± s.d., n = 3 technical replicates). * denotes P = 0.012 (two-sided t-test). No significancy (P = 0.395) between K+ + Mg2+ and K+ + Mn2+ (two-sided t-test). o, Circular dichroism thermal analysis of Lettuce and Bibb Lettuce in the presence and absence of DFHBI1T at 290 nm.

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Extended Data Fig. 6 R-loop characterization, compactness study of RNA and DNA, and structure and functions of DNA and RNA aptamers.

a, Autoradiogram of 6% non-denaturing PAGE demonstrating R-loop formation (arrow) showed on Fig. 4. For uncropped gel, see Supplementary Fig. 1. The detection of R-loop formation experiment by non-denaturing PAGE was replicated three times. b, Schematic of 260-nt R-loop assay. c, Fluorescence analysis of longer R-loop formation as depicted on b. Fluorescence of DFHBI-1T in the presence of buffer, RNA, 310-bp dsDNA, dsDNA + RNA not co-annealed, dsDNA + RNA co-annealed, normalized to wild-type Lettuce (mean ± s.d., n = 3 technical replicates). d, RNA and DNA compactness study plotted graph using data from Supplementary Table 1. e, Lettuce–DFHBI-1T co-crystal structure (this study). f, NMR structure of the AMP-binding DNA aptamer in complex with two AMP molecules (ref. 71; PDB:1AW4). g, NMR structure of a DNA aptamer bound to argininamide (ref. 72; PDB:1DB6). h, NMR structure of the OBA3 DNA aptamer bound to ochratoxin A (ref. 73; PDB:6J2W). i, NMR structure of the OBA36 DNA aptamer bound to ochratoxin A (ref. 74; PDB:7W9N). j, Fluorescence of wild-type Lettuce used in the crystallographic studies, Split Lettuce, Lettuce with shorter P1 used in fluorescence studies (Supplementary Table 3), RNA comprised of the Lettuce sequence, and split Lettuce comprised by DNA (34 nts) and RNA (16 nts of 3' terminus) in the presence of DFHBI-1T (mean ± s.d., n = 3 technical replicates). Asterisk denotes P = 0.002 (two-sided t-test). No significant difference (P= 0.407) between Lettuce w.t. and Lettuce with shorter P1 (two-sided t-test). k, Pucker angles of each deoxynucleotide of Lettuce–fluorophore complexes (mean of 8 structures). l, Pucker angles of each deoxynucleotide of the RNA-ligating deoxyribozyme 9DB1 (ref. 49; PDB:5CKK). Puckers of core deoxynucleotides are in magenta; puckers of deoxynucleotides that hybridize to the RNA product strand are in cyan. m, Pucker angles of each deoxynucleotide of the RNA-cleaving deoxyribozyme Dz36 (ref. 50; PDB:5XM8). Puckers of core deoxynucleotides are in orange; puckers of deoxynucleotides that hybridize to the RNA substrate strand are in gray. n, Pucker angles for each ribose of the RNA fluorogenic aptamer iSpinach in complex with DFHBI (ref. 48; PDB:5OB3). Individual puckers for all four aptamers are in Supplementary Table 2.

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Extended Data Table 1 Crystallographic statistics
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Extended Data Table 2 Crystallographic statistics
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Extended Data Table 3 Cryo-EM data collection and processing parameters for unliganded Lettuce
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Passalacqua, L.F.M., Banco, M.T., Moon, J.D. et al. Intricate 3D architecture of a DNA mimic of GFP. Nature 618, 1078–1084 (2023). https://doi.org/10.1038/s41586-023-06229-8

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