Theophylline aptamer

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Timeline

1994

Aptamers are isolated out for the first time[1]

1997

First demonstrated that the aptamer could be linked to stem II of a self-cleaving hammerhead ribozyme[2]

1997

Determination of RNA structural interactions in the theophylline ligand binding site using NMR techniques[3]

1999

First discovery of theophylline-reactive riboswitches for controlling gene expression in vivo[4]

2001

Created a binary RNA switch by linking the theophylline and FMN aptamers to the hammerhead[5]

2005

Demonstrated that the aptamer can be truncated to a 13-mer aptamer and still retain selective binding[6]

2005

Theophylline-controlled 'anti-switch' found to control green fluorescent protein expression in Saccharomyces cerevisiae[7]

2006

Young and Deiters added a second level of control by photocaging the theophylline ligand, resulting in a photochemical activation of the ribozyme by liberation of the caging group[8]

2007

Developed a computational model to generate functional theophylline ribozymes[9]

2009

A flow cytometry-based screen for identifying synthetic riboswitches that induce robust increases in gene expression in the presence of theophylline[10]

2013

Using a computational model, Penchovsky developed and tested a theophyllinehammerhead ribozyme fusion that was inactivated with a 30-fold dynamic range[11]

2013

Developed three OFF theophylline switches that are functional in vivo [12]

2015

The aptamers found to optimise ribose enlightenment[13]

2015

The better standardization of gene expression or cellular processes[14]

2015

Six new theophylline aptamers discovered using computerised screening[15]

2018

The aptamers can build logic gates in cells[16]

2018

New theophylline-reactive aptamers are screened from a synthetic library in Escherichia coli[17]

2022

The structure of the aptamer, both in isolation and in complex with theophylline and the four best hits, was determined by X-ray crystallography[30]

Description

In 1994, Polisky, B. et al. used SELEX to screen for high-affinity RNA aptamers with theophylline-binding solution structures. In 1997, NMR techniques visualised the interaction of RNA structures in the ligand-binding site. In 1998, it was found that U27 and G27 RNAs bound theophylline with low-affinity (Kd values > 4 μM). NMR spectroscopy of U27 RNA revealed an A7-U27 base pair in the free RNA, which prevented the formation of a key structural motif in the critical base platform and thus inhibited theophylline binding. In 2022, the crystal structure of the theophylline aptamer was resolved[1,3,4,30].



SELEX

In 1994, Polisky, B et al. used SELEX to identify RNA molecules with an affinity for theophylline. They created a pool of 1014 RNA molecules, each comprising a 40-nucleotide region of random sequence. The RNA pool was then introduced to a Sepharose column that had 1-carboxypropyl theophylline covalently cross-linked to it. The bound RNA was subsequently eluted by adding 0.1 M theophylline. The eluted RNA was then converted to DNA and amplified via polymerase chain reaction (PCR), following the described procedure[1].

Detailed information are accessible on SELEX page.



Structure

2D representation

In 1997, Pardi.A. et al. obtained multiple different RNA sequences through in vitro selection procedures, including the SELEX technique, and designed a high-affinity RNA aptamer after comparing the information of these sequences. The RNA aptamer is characterised by a sequence that forms a distinct secondary structure, as illustrated in the subsequent diagrams. Here we used RiboDraw to complete the figure, based the 3D structure information[3].

5'-GGCAUACCAGCCGAAAGGCCCUUGGCAGCGUC-3'

drawing

3D visualisation

In 1997, Pardi.A. et al. used NMR and X-PLOR 3.113 calculations to demonstrate the solution structure of a high-affinity RNA-theophylline complex, where the C27 nucleotide was identified as the key residue for recognising theophylline and discriminating it from caffeine. The PDB ID for the NMR structure of the RNA-theophylline complex is 1EHT. In 2022, Rogers.J. et al. determined the crystal structure of an RNA aptamer-theophylline complex. Owing to the high similarity between the solved NMR 3D structure and the following crystal 3D structure, only the crystal 3D structure is presented here. The PDB ID for the crystal structure of the theophylline aptamer is 8D28[3,30].

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

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

drawing PDBe Molstar




Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 8D28 by X-ray Crystallography. Theophylline (shown in sticks) is labeled in magenta. Right: The hydrogen bonds of binding sites of the aptamer bound with Theophylline.

drawing drawing


Ligand information

SELEX ligand

To determine the dissociation constants (Kd) of the theophylline aptamer and its ligands, the RNA was synthesised with a Cy5 fluorophore at the 3' end. Strand invasion assays were conducted by incubating the labelled aptamer with a complementary oligonucleotide carrying a fluorescence quencher. The fluorescence changes were measured upon the addition of various compounds, and the data were fitted to determine the IC50 values, which served as an estimate of Kd. Surface plasmon resonance (SPR) was used for precise Kd determination, where the RNA aptamer was immobilised on a CM5 sensor chip via biotin and NeutrAvidin. The ligands were injected at varying concentrations, and the binding data were analysed to obtain Kd values. The compounds TAL1, TAL2, TAL3, and TAL4 were found to have Kd values of 0.0020, 0.0091, 0.023, and 0.28 µM, respectively, demonstrating higher affinity than theophylline (0.67 µM). Theophylline aptamer ligand 1 (TAL1), is an aminomethyl-substituted pteridinone that has a similar molecular weight as that of theophylline. The other three (TAL2, TAL3, and TAL4) have higher molecular weights, with differing substitutions of a common quinazolinone ring system[30].

drawing

Structure ligand

A methylxanthine derivative from tea with diuretic, smooth muscle relaxant, bronchial dilation, cardiac and central nervous system stimulant activities. Mechanistically, theophylline acts as a phosphodiesterase inhibitor, adenosine receptor blocker, and histone deacetylase activator. Theophylline is marketed under several brand names such as Uniphyl and Theochron, and it is indicated mainly for asthma, bronchospasm, and COPD.-----FromDrugbank

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
Theophylline 2153 C7H8N4O2 180.16 g/mol 58-55-9 7360mg/L (at 25 °C) DB00277
drawing drawing

Similar compound(s)

We screened the compounds with great similarity totheophylline 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
ZINC18043251 Theophylline 58-55-9 2153 drawing
ZINC13517144 1-Methylxanthine 6136-37-4 80220 drawing
ZINC4685854 3-Methylxanthine 1076-22-8 70639 drawing
ZINC100018165 8-Chlorotheophylline 85-18-7 10661 drawing
ZINC100005670 8-Bromotheophylline 10381-75-6 11808 drawing
ZINC1084 caffeine 21399 2519 drawing
ZINC8616085 3-Methylguanine 2958-98-7 76292 drawing
ZINC100043983 1,3-Dimethyluric acid 944-73-0 70346 drawing
ZINC403604 Isocaffeine 519-32-4 1326 drawing


References

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