DIR2s-apt aptamer

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Timeline

2017

An RNA aptamer selected for binding to the fluorogenic cyanine dye dimethylindole red (DIR), also binds oxazole thiazole blue (OTB)[1]

2018

The crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution respectively was solved[2]

Description

In 2017, Bruce A. Armitage and Xiaohong Tan et al. screened out aptamer, which has affinity to both dimethylindole red and oxazole thiazole blue and could give two well-resolved emission colors. In 2018, Joseph A. Piccirilli and Sandip A. Shelke et al. solved the overall structure of OTB-SO3-bound DIR2s aptamer-Fab complex by collecting Small Angle X-ray Scattering data of crystals[1,2].



SELEX

Biotinylated DIR was used as Positive target to facilitate the enrichment process. The RNA pool used for this selection contained ~1014 unique sequences and universal, stem-loop forming sequence flanked by randomized region. RNA pool was asymmetric, with the putative stem-loop shifted toward the 5'-end of the sequence. The aptamer from the asymmetric pool was abundantly represented after 14 rounds of selection. After sequence minimization and optimization, a 57 nucleotide anti-DIR RNA aptamer (DIR2s-apt) which can bind and activate DIR fluorescence was obtained[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

The RNA aptamer construct adopts an elongated structure composed of four helices (P1, P2, P3, and P4) and three loops (L1, L3, and the Fab BL3-6 binding loop, L2). The stem-loop P4-L3 coaxially stacks on the helix P1 while loop L1 and its associated short stem P2 orients parallel to and interacts with the loop L3 through base pairing and tertiary interactions. Here we used ribodraw to complete the figure, through the 3D structure information[1].

5'-GGAUGCGCCUUGAAAAGCCUGCGAAACACGCAGCUGGUGAAUGACAGCUAUGGCGCAUCC-3'

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3D visualisation

In 2018, Joseph A. Piccirilli et al. solved the overall structure of OTB-SO3 fluorophore-bound DIR2s aptamer-Fab complex at 2.0 Å and DIR2s aptamer structures in the presence and absence of the OTB-SO3 fluorophore at 1.8 Å by collecting small angle X-ray scattering data of crystals. RNA Mango adopts a mixed parallel/antiparallel G-quadruplex architecture, stabilised by six loops that interconnect the 12 guanine residues of its three G-quartets. A22, located within a propeller loop bridging T3 and T1, contributes to the formation of a pentad by engaging the sugar edge of G18 via its Hoogsteen face. U15, A20, and A25-each forming one of three directional reversal loops positioned above T3-collectively establish the TO1-Biotin-binding site through direct structural coordination. This structural arrangement is stabilised by extensive interactions between loops L1 and L3. The PDB ID of the two structures are 6DB8 (1.87 Å) and 6DB9 (2.025 Å)[2].

Additional available structures that have been solved and detailed information are accessible on Structures page.

(Clicking the "Settings/Controls info" to turn Spin off)      

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Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 6DB8 by NMR and collecting small angle X-ray scattering data of crystals. OTB-SO3 (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with OTB-SO3 or other nucleotides surround small molecules.

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Ligand information

SELEX ligand

Binding affinity was determined by fluorescence titration in the buffer and calculated by the concentration correlation function between aptamer concentration and fluorescence intensity[1].

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Structure ligand

OTB (3-methyl-2-[(3-methyl-1,3-benzothiazol-3-ium-2-yl)methylidene]-1,3-benzoxazole) is a blue-emitting fluorogen which belongs to fluorogenic cyanine dye.-----Journal of the American Chemical Society, 139(26), 9001–9009.

PubChem CID: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

Drugbank: a comprehensive database with detailed information on drugs and drug targets.

Name PubChem CID Molecular Formula MW CAS Solubility Drugbank ID
OTB 54402972 C17H15N2OS+ 295.4 g/mol NA NA NA
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Similar compound(s)

We screened the compounds with great similarity to OTB by using the ZINC database and showed some of the compounds' structure diagrams. For some CAS numbers not available, we will supplement them with Pubchem CID.

ZINC ID: a compound identifier used by the ZINC database, one of the largest repositories for virtual screening of drug-like molecules.

PubChem CID: a unique identifier for substances in the PubChem database.

CAS number: a global registry number for chemical substances.

ZINC ID Name CAS Pubchem CID Structure
ZINC2509998 NA NA 17729 drawing
ZINC20070324 NA NA 22332836 drawing
ZINC844804 NA NA 1110831 drawing
ZINC107280752 NA NA 54298998 drawing
ZINC32105922 NA NA 12690696 drawing
ZINC272646 NA NA 779797 drawing
ZINC100399021 NA NA 5816761 drawing
ZINC100141140 NA NA 6432295 drawing
ZINC169438545 NA NA 154540469 drawing


References

[1] Fluoromodules consisting of a promiscuous RNA aptamer and red or blue fluorogenic cyanine dyes: Selection, characterization, and bioimaging.
Tan, X., Constantin, T. P., Sloane, K. L., Waggoner, A. S., Bruchez, M. P., & Armitage, B. A.
Journal of the American Chemical Society, 139(26), 9001–9009. (2017)
[2] Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.
Shelke, S. A., Shao, Y., Laski, A., Koirala, D., Weissman, B. P., Fuller, J. R., Tan, X., Constantin, T. P., Waggoner, A. S., Bruchez, M. P., Armitage, B. A., & Piccirilli, J. A.
Nature communications, 9(1), 4542. (2018)