Tobramycin aptamer

Apr 24, 2024 • Linfei Li, Ke Chen

Timeline

Description

Tobramycin aptamers, a class of high-affinity RNA molecules obtained through in vitro selection, were found to exhibit specific recognition of the aminoglycoside antibiotic tobramycin. Research demonstrated that these aptamers formed binding pockets via distinctive stem-loop structures. These structures adopted a “zipper-like” fold, which broadened the RNA’s major groove geometry through the synergistic arrangement of mismatched base pairs (e.g., G•U, U•U) and Watson-Crick base pairs (e.g., U•A). Tobramycin was positioned within the RNA’s major groove, partially encapsulated between the groove’s base and protruding bases (e.g., G15 or C15), forming a semi-enclosed chamber. Its binding was stabilised by hydrogen bonding and electrostatic interactions. For instance, the amino groups (NH₃⁺) of tobramycin could form hydrogen-bond networks with RNA base edges (e.g., N7, O4) or the phosphate backbone, while its aminosugar ring III enhanced binding through hydrophobic interactions with non-polar pyrimidine rings. Selection experiments revealed that high-affinity aptamers (with Kd values in the nM range) possessed highly conserved sequences. Their core binding domains were concentrated within predicted stem-loop regions, with affinity directly correlated to stem-loop stability. Quantitative analyses using fluorescently labelled tobramycin analogues (e.g., PYT) indicated that aptamers exhibited cross-binding capability with structurally similar aminoglycosides (e.g., neomycin), but no significant interaction with non-aminoglycoside antibiotics (e.g., erythromycin), underscoring their specificity. These studies not only elucidated the molecular mechanisms underlying RNA-aminoglycoside interactions but also provided critical insights for antibiotic optimisation and the design of novel RNA-targeted therapeutics informed by structural data^[1,2,3].

SELEX

An RNA diversity library was employed to select sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound specifically to tobramycin. The selected RNA was then reverse-transcribed into DNA and subsequently cloned. At low selection stringency, an extremely large number of clones, on the order of 10⁷, produced RNAs capable of binding tobramycin with dissociation constants (Kd) in the µM range. These values were similar to those observed for the binding of tobramycin to Escherichia coli ribosomes. Sequencing of 18 of these clones revealed no obvious consensus sequence. However, at higher selection stringencies (with Kd values in the nM range), only two consensus sequences for binding were observed^[1].

Detailed information are accessible on SELEX page.

Structure

2D representation

Here we used ribodraw to complete the figure, through the 3D structure information^[2,3].

5'-GGCACGAGGUUUAGCUACACUCGUGCC-3'

5'-ACUUGGUUUAGGUAAUGAGU-3'

3D visualisation

Patel, D. J. et al. described a solution-structure determination of the tobramycin-RNA aptamer I complex, obtained using nuclear magnetic resonance (NMR) and molecular dynamics. The PDB ID of this structure is 1TOB^[2].

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

Patel, D. J. et al. described the structure of the complex was calculated by NMR and the X-PLOR programme, and molecular dynamics constraints were performed to demonstrate the solution structure of the complex of the aminoglycoside antibiotic tobramycin in conjunction with stem-loop RNA aptamer II. The PDB ID of this structure is 2TOB^[3].

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

Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 1TOB by NMR. TOB (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with TOB.

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 2TOB by NMR. TOB (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with TOB.

Ligand information

SELEX ligand

Rando, R. R. et al. used a variety of methods, including cloned RNA fluorescence, equilibrium gel filtration, and polymerase chain reaction (PCR) to determine the affinity of tobramycin RNA aptamer in solution^[1].

Structure ligand

Tobramycin is an aminoglycoside antibiotic used to treat cystic fibrosis-associated bacterial, lower respiratory tract, urinary tract, eye, skin, bone, and skin structure infections.-----From Drugbank

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	Molecular Weight	CAS	Solubility	Drugbank ID
Tobramycin	36294	C18H37N5O9	467.5 g/mol	32986-56-4	5.37e+01 g/L	DB00684

Similar compound(s)

We screened the compounds with great similarity toTobramycin 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
ZINC8551162	(2R,3S,4R,5S,6S)-4-amino-2-[(1R,2R,3R,4S,6R)-4,6-diamino-3-[(2S,3S,5S,6S)-3-amino-6-(aminomethyl)-5-hydroxyoxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-6-(hydroxymethyl)oxane-3,5-dio	NA	92331686
ZINC8551164	(2R,3S,4R,5S,6S)-4-amino-2-[(1R,2R,3R,4S,6R)-4,6-diamino-3-[(2R,3S,5S,6S)-3-amino-6-(aminomethyl)-5-hydroxyoxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-6-(hydroxymethyl)oxane-3,5-diol	NA	92331688
ZINC8551165	(2R,3S,4R,5S,6S)-4-amino-2-[(1R,2R,3R,4S,6R)-4,6-diamino-3-[(2R,3S,5S,6R)-3-amino-6-(aminomethyl)-5-hydroxyoxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-6-(hydroxymethyl)oxane-3,5-diol	NA	92331689
ZINC8551163	(2R,3S,4R,5S,6S)-4-amino-2-[(1R,2R,3R,4S,6R)-4,6-diamino-3-[(2S,3S,5S,6R)-3-amino-6-(aminomethyl)-5-hydroxyoxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-6-(hydroxymethyl)oxane-3,5-diol	NA	92331687
ZINC8214692	Tobramycin	32986-56-4	36294
ZINC43562055	(2S,3R,4R,5S,6S)-2-(Aminomethyl)-6-[(1R,2R,3S,4R,6S)-4,6-diamino-3-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxyoxane-3,4,5-triol	NA	11274101
ZINC8214590	Kanamycin	59-01-8	6032
ZINC53132258	Bekanamycin	4696-76-8	439318
ZINC8214383	Dibekacin	34493-98-6	470999

References

[1] Specific binding of aminoglycoside antibiotics to RNA.
Wang, Y., & Rando, R. R.
Chemistry & biology, 2(5), 281–290. (1995)
[2] Accharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex.
Jiang, L., Suri, A. K., Fiala, R., & Patel, D. J.
SChemistry & biology, 4(1), 35–50. (1997)
[3] Solution structure of the tobramycin-RNA aptamer complex.
Jiang, L., & Patel, D. J.
Nature structural biology, 5(9), 769–774. (1998)
[4] Tobramycin affinity tag purification of spliceosomes.
Hartmuth, K., Vornlocher, H. P., & Lührmann, R.
Methods in molecular biology (Clifton, N.J.), 257, 47–64. (2004)
[5] Electrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity.
Keller, K. M., Breeden, M. M., Zhang, J., Ellington, A. D., & Brodbelt, J. S.
Journal of mass spectrometry : JMS, 40(10), 1327–1337. (2005)
[6] Achieving reproducible performance of electrochemical, folding aptamer-based sensors on microelectrodes: challenges and prospects.
Liu, J., Wagan, S., Dávila Morris, M., Taylor, J., & White, R. J.
Analytical chemistry, 86(22), 11417–11424. (2014)
[7] Rationally designing aptamer sequences with reduced affinity for controlled sensor performance.
Schoukroun-Barnes, L. R., & White, R. J.
Sensors (Basel, Switzerland), 15(4), 7754–7767. (2015)
[8] Gold nanoparticle based photometric determination of tobramycin by using new specific DNA aptamers.
Han, X., Zhang, Y., Nie, J., Zhao, S., Tian, Y., & Zhou, N.
Mikrochimica acta, 185(1), 4. (2017)
[9] Aptamer-enabled uptake of small molecule ligands.
Auwardt, S. L., Seo, Y. J., Ilgu, M., Ray, J., Feldges, R. R., Shubham, S., Bendickson, L., Levine, H. A., & Nilsen-Hamilton, M.
Scientific reports, 8(1), 15712. (2018)
[10] Electrochemical detection of tobramycin based on enzymes-assisted dual signal amplification by using a novel truncated aptamer with high affinity.
Nie, J., Yuan, L., Jin, K., Han, X., Tian, Y., & Zhou, N.
Biosensors & bioelectronics, 122, 254–262. (2018)
[11] Creening, Post-SELEX Optimization and Application of DNA Aptamers Specific for Tobramycin.
Zhou, N., Cai, R., & Han, X. S.
Methods in molecular biology (Clifton, N.J.), 2070, 1–18 (2020)
[12] Structure-switching aptamer triggering signal amplification strategy for tobramycin detection based on hybridization chain reaction and fluorescence synergism.
Wang, J., Li, H., Du, C., Li, Y., Ma, X., Yang, C., Xu, W., & Sun, C.
Talanta, 243, 123318. (2022)
[13] Development of a novel tobramycin dependent riboswitch.
Kraus, L., Duchardt-Ferner, E., Bräuchle, E., Fürbacher, S., Kelvin, D., Marx, H., Boussebayle, A., Maurer, L. M., Bofill-Bosch, C., Wöhnert, J., & Suess, B.
Nucleic acids research, 51(20), 11375–11385. (2023)