Signaling Aptamers with specific target binding properties

Designing oligonucleotides such as aptamers with specific target binding properties widens the application range of nucleic acid-based fluorescent probes for detecting many analytes, including small molecules, proteins, nucleic acids, ions, and whole cells.

Aptamer probes that exploit non-nucleic acid analytes' selective recognition allow the design of immunosensors. The design and construction of hybridization and aptamer probes are similar. Reporter groups covalently attached to oligonucleotides (DNA or RNA) with predefined sequences enable the design of fluorescence-based aptamer probes.

Fluorescent labels act as transducers, transforming biorecognition such as hybridization or ligand binding into a fluorescence signal. Fluorescent labels have several advantages when compared to radioactive labels, including high sensitivity and multiple transduction approaches such as fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching.

Two types of fluorophores enable the labeling of oligonucleotides:

1.    Dyes that change their fluorescence properties when binding nucleic acids are used in single-labeled probes.

2.    Fluorophores with strong fluorescence that change their emission intensity when brought into contact with each other or with a quencher molecule. Examples are fluorescein and rhodamine dyes.

Standard detection modes are fluorescence resonance energy transfer, excimer–monomer switching, hybridization probes, binary probes, and molecular beacons.

Figure 1: Schematics of a fluorescent biosensor.

Oligonucleotides with defined sequences and structures can recognize specific non-nucleic acid targets - these single-stranded nucleic acid sequences obtained by the SELEX method are known as aptamers.

Aptamers with sequences characteristic of specific protein-binding regions enable protein recognition.

The anti-thrombin aptamer is an example. A 15-mer oligonucleotide, with the sequence 5′-d(GGTTGGTGTGGTTGG)-3′, forms an intramolecular quadruplex with two G-tetrads firmly binding to thrombin.

Tan and co-workers have used the pyrene excimer monomer switching (EMS) approach to design a wavelength-shifting aptamer probe.

The probe contains a platelet-derived growth factor (PDGF)-binding DNA aptamer labeled with two pyrene molecules.

In this aptamer, the two ends are far away in the absence of PDGF but close to each other in the presence of the target.

When bound to PDGF, the aptamer switches its fluorescence emission from 400 nm (pyrene monomer, with a fluorescence lifetime of ∼5 ns) to 485 nm (pyrene excimer, with a lifetime of ∼40 ns).

The wavelength-shifting and time-resolved measurement characteristics allow for sensing target molecules in complex sample matrixes.

Sensing is even possible in cell media, in which strong background fluorescence often makes intensity-based detection difficult.

Chen recently reviewed recent advances in developing excimer-based fluorescence probes useful for biological applications.

Reference

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