6C63

Crystal Structure of the Mango-II Fluorescent Aptamer Bound to TO1-Biotin


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.90 Å
  • R-Value Free: 0.230 
  • R-Value Work: 0.185 
  • R-Value Observed: 0.190 

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This is version 1.1 of the entry. See complete history


Literature

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

Trachman 3rd., R.J.Abdolahzadeh, A.Andreoni, A.Cojocaru, R.Knutson, J.R.Ryckelynck, M.Unrau, P.J.Ferre-D'Amare, A.R.

(2018) Biochemistry 57: 3544-3548

  • DOI: https://doi.org/10.1021/acs.biochem.8b00399
  • Primary Citation of Related Structures:  
    6C63, 6C64, 6C65

  • PubMed Abstract: 

    Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.


  • Organizational Affiliation

    Biochemistry and Biophysics Center , National Heart, Lung and Blood Institute , 50 South Drive, MSC 8012 , Bethesda , Maryland 20892-8012 , United States.


Macromolecules
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Entity ID: 1
MoleculeChains LengthOrganismImage
RNA (36-MER)
A, B
36synthetic construct
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains LengthOrganismImage
RNA (32-MER)32synthetic construct
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
EKJ
Query on EKJ

Download Ideal Coordinates CCD File 
D [auth A],
H [auth B],
K [auth C]
4-[(3-{2-[(2-methoxyethyl)amino]-2-oxoethyl}-1,3-benzothiazol-3-ium-2-yl)methyl]-1-methylquinolin-1-ium
C23 H25 N3 O2 S
WEFGJTSJMBBARG-UHFFFAOYSA-O
K
Query on K

Download Ideal Coordinates CCD File 
E [auth A]
F [auth A]
G [auth A]
I [auth B]
J [auth B]
E [auth A],
F [auth A],
G [auth A],
I [auth B],
J [auth B],
L [auth C],
M [auth C],
N [auth C]
POTASSIUM ION
K
NPYPAHLBTDXSSS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.90 Å
  • R-Value Free: 0.230 
  • R-Value Work: 0.185 
  • R-Value Observed: 0.190 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 36.831α = 90
b = 182.412β = 90
c = 107.494γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data reduction
SCALEPACKdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2018-08-08
    Type: Initial release
  • Version 1.1: 2023-10-04
    Changes: Data collection, Database references, Derived calculations, Refinement description