Summary information [schematics · tetrads · helices · stems · costacks · homepage]

PDB-id
6c64
Class
RNA
Method
X-ray (3.0 Å)
Summary
Crystal structure of the mango-ii fluorescent aptamer bound to to3-biotin
Reference
Trachman 3rd., R.J., Abdolahzadeh, A., Andreoni, A., Cojocaru, R., Knutson, J.R., Ryckelynck, M., Unrau, P.J., Ferre-D'Amare, A.R.: (2018) "Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness." Biochemistry, 57, 3544-3548.
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.
G4 notes
9 G-tetrads, 2 G4 helices, 3 G4 stems, 1 G4 coaxial stack · 2(-P-P-P), parallel(4+0), UUUU · coaxial interfaces: 5'/5'

Base-block schematics in six views [summary · tetrads · helices · stems · costacks · homepage]

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List of 9 G-tetrads [summary · schematics · helices · stems · costacks · homepage]

 1 glyco-bond=---- groove=---- planarity=0.221 type=other  nts=4 GGGG A.G10,A.G15,A.G20,A.G26
 2 glyco-bond=---- groove=---- planarity=0.138 type=planar nts=4 GGGG A.G11,A.G16,A.G21,A.G27
 3 glyco-bond=---- groove=---- planarity=0.067 type=planar nts=4 GGGG A.G13,A.G29,A.G24,A.G18
 4 glyco-bond=---- groove=---- planarity=0.305 type=bowl-2 nts=4 GGGG B.G10,B.G15,B.G20,B.G26
 5 glyco-bond=---- groove=---- planarity=0.101 type=planar nts=4 GGGG B.G11,B.G16,B.G21,B.G27
 6 glyco-bond=---- groove=---- planarity=0.118 type=planar nts=4 GGGG B.G13,B.G29,B.G24,B.G18
 7 glyco-bond=---- groove=---- planarity=0.314 type=bowl-2 nts=4 GGGG D.G10,D.G15,D.G20,D.G26
 8 glyco-bond=---- groove=---- planarity=0.092 type=planar nts=4 GGGG D.G11,D.G16,D.G21,D.G27
 9 glyco-bond=---- groove=---- planarity=0.108 type=planar nts=4 GGGG D.G13,D.G29,D.G24,D.G18

List of 2 G4-helices [summary · schematics · tetrads · stems · costacks · homepage]

In DSSR, a G4-helix is defined by stacking interactions of G-tetrads, regardless of backbone connectivity, and may contain more than one G4-stem.

Helix#1, 3 G-tetrad layers, INTRA-molecular, with 1 stem

 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G10,A.G15,A.G20,A.G26
 2  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G11,A.G16,A.G21,A.G27
 3  glyco-bond=---- groove=---- Major-->WC nts=4 GGGG A.G13,A.G18,A.G24,A.G29
  step#1  pm(>>,forward)  area=8.81  rise=3.31 twist=34.2
  step#2  pp(><,inward)   area=28.14 rise=3.39 twist=27.3
  strand#1 RNA glyco-bond=--- nts=3 GGG A.G10,A.G11,A.G13
  strand#2 RNA glyco-bond=--- nts=3 GGG A.G15,A.G16,A.G18
  strand#3 RNA glyco-bond=--- nts=3 GGG A.G20,A.G21,A.G24
  strand#4 RNA glyco-bond=--- nts=3 GGG A.G26,A.G27,A.G29

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2 stacking diagrams
 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G10,A.G15,A.G20,A.G26
2 glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G11,A.G16,A.G21,A.G27
step#1 pm(>>,forward) area=8.81 rise=3.31 twist=34.2

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 2  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G11,A.G16,A.G21,A.G27
3 glyco-bond=---- groove=---- Major-->WC nts=4 GGGG A.G13,A.G18,A.G24,A.G29
step#2 pp(><,inward) area=28.14 rise=3.39 twist=27.3

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Helix#2, 6 G-tetrad layers, inter-molecular, with 2 stems

 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG B.G13,B.G29,B.G24,B.G18
 2  glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G11,B.G27,B.G21,B.G16
 3* glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G10,B.G26,B.G20,B.G15
 4  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G15,D.G20,D.G26,D.G10
 5  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G16,D.G21,D.G27,D.G11
 6  glyco-bond=---- groove=---- Major-->WC nts=4 GGGG D.G18,D.G24,D.G29,D.G13
  step#1  pp(><,inward)   area=27.88 rise=3.38 twist=28.2
  step#2  mp(<<,backward) area=8.23  rise=3.29 twist=34.1
  step#3  mm(<>,outward)  area=22.61 rise=3.53 twist=8.8
  step#4  pm(>>,forward)  area=8.81  rise=3.31 twist=33.3
  step#5  pp(><,inward)   area=26.04 rise=3.34 twist=30.0
  strand#1 RNA glyco-bond=------ nts=6 GGGGGG B.G13,B.G11,B.G10,D.G15,D.G16,D.G18
  strand#2 RNA glyco-bond=------ nts=6 GGGGGG B.G29,B.G27,B.G26,D.G20,D.G21,D.G24
  strand#3 RNA glyco-bond=------ nts=6 GGGGGG B.G24,B.G21,B.G20,D.G26,D.G27,D.G29
  strand#4 RNA glyco-bond=------ nts=6 GGGGGG B.G18,B.G16,B.G15,D.G10,D.G11,D.G13

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5 stacking diagrams
 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG B.G13,B.G29,B.G24,B.G18
2 glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G11,B.G27,B.G21,B.G16
step#1 pp(><,inward) area=27.88 rise=3.38 twist=28.2

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 2  glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G11,B.G27,B.G21,B.G16
3* glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G10,B.G26,B.G20,B.G15
step#2 mp(<<,backward) area=8.23 rise=3.29 twist=34.1

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 3* glyco-bond=---- groove=---- Major-->WC nts=4 GGGG B.G10,B.G26,B.G20,B.G15
4 glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G15,D.G20,D.G26,D.G10
step#3 mm(<>,outward) area=22.61 rise=3.53 twist=8.8

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 4  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G15,D.G20,D.G26,D.G10
5 glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G16,D.G21,D.G27,D.G11
step#4 pm(>>,forward) area=8.81 rise=3.31 twist=33.3

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 5  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G16,D.G21,D.G27,D.G11
6 glyco-bond=---- groove=---- Major-->WC nts=4 GGGG D.G18,D.G24,D.G29,D.G13
step#5 pp(><,inward) area=26.04 rise=3.34 twist=30.0

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List of 3 G4-stems [summary · schematics · tetrads · helices · costacks · homepage]

In DSSR, a G4-stem is defined as a G4-helix with backbone connectivity. Bulges are also allowed along each of the four strands.

Stem#1, 2 G-tetrad layers, 3 loops, INTRA-molecular, UUUU, parallel, 2(-P-P-P), parallel(4+0)

 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G10,A.G15,A.G20,A.G26
 2  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG A.G11,A.G16,A.G21,A.G27
  step#1  pm(>>,forward)  area=8.81  rise=3.31 twist=34.2
  strand#1  U RNA glyco-bond=-- nts=2 GG A.G10,A.G11
  strand#2  U RNA glyco-bond=-- nts=2 GG A.G15,A.G16
  strand#3  U RNA glyco-bond=-- nts=2 GG A.G20,A.G21
  strand#4  U RNA glyco-bond=-- nts=2 GG A.G26,A.G27
  loop#1 type=propeller strands=[#1,#2] nts=3 AGA A.A12,A.G13,A.A14
  loop#2 type=propeller strands=[#2,#3] nts=3 AGA A.A17,A.G18,A.A19
  loop#3 type=propeller strands=[#3,#4] nts=4 AAGA A.A22,A.A23,A.G24,A.A25

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Stem#2, 2 G-tetrad layers, 3 loops, INTRA-molecular, UUUU, parallel, 2(-P-P-P), parallel(4+0)

 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG B.G10,B.G15,B.G20,B.G26
 2  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG B.G11,B.G16,B.G21,B.G27
  step#1  pm(>>,forward)  area=8.23  rise=3.29 twist=34.1
  strand#1  U RNA glyco-bond=-- nts=2 GG B.G10,B.G11
  strand#2  U RNA glyco-bond=-- nts=2 GG B.G15,B.G16
  strand#3  U RNA glyco-bond=-- nts=2 GG B.G20,B.G21
  strand#4  U RNA glyco-bond=-- nts=2 GG B.G26,B.G27
  loop#1 type=propeller strands=[#1,#2] nts=3 AGA B.A12,B.G13,B.A14
  loop#2 type=propeller strands=[#2,#3] nts=3 AGA B.A17,B.G18,B.A19
  loop#3 type=propeller strands=[#3,#4] nts=4 AAGA B.A22,B.A23,B.G24,B.A25

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Stem#3, 2 G-tetrad layers, 3 loops, INTRA-molecular, UUUU, parallel, 2(-P-P-P), parallel(4+0)

 1  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G10,D.G15,D.G20,D.G26
 2  glyco-bond=---- groove=---- WC-->Major nts=4 GGGG D.G11,D.G16,D.G21,D.G27
  step#1  pm(>>,forward)  area=8.81  rise=3.31 twist=33.3
  strand#1  U RNA glyco-bond=-- nts=2 GG D.G10,D.G11
  strand#2  U RNA glyco-bond=-- nts=2 GG D.G15,D.G16
  strand#3  U RNA glyco-bond=-- nts=2 GG D.G20,D.G21
  strand#4  U RNA glyco-bond=-- nts=2 GG D.G26,D.G27
  loop#1 type=propeller strands=[#1,#2] nts=3 AGA D.A12,D.G13,D.A14
  loop#2 type=propeller strands=[#2,#3] nts=3 AGA D.A17,D.G18,D.A19
  loop#3 type=propeller strands=[#3,#4] nts=4 AAGA D.A22,D.A23,D.G24,D.A25

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List of 1 G4 coaxial stack [summary · schematics · tetrads · helices · stems · homepage]

 1 G4 helix#2 contains 2 G4 stems: [#2,#3]  [5'/5']

List of 0 non-stem G4-loops (including the two closing Gs)