Aptamers Embedded in Polyacrylamide Nanoparticles: A Tool for in Vivo Metabolite Sensing

Abstract

We describe a new type of aptamer-based optical nanosensor which uses the embedding of target responsive oligonucleotides in porous polyacrylamide nanoparticles to eliminate nuclease instability. The latter is a common problem in the use of aptamer sensors in biological environments. These aptamers embedded in nanoparticles (AptaNPs) are proposed as a tool in real-time metabolite measurements in living cells. The AptaNPs comprise 30 nm polyacrylamide nanoparticles, prepared by inverse microemulsion polymerization, which contain water-soluble aptamer switch probes (ASPs) trapped in the porous matrix of the nanoparticles. The matrix acts as a molecular fence allowing rapid diffusion of small metabolites into the particles to interact with the aptamer molecules, but at the same time it retains the larger aptamer molecules inside the nanoparticles providing protection against intracellular degradation. We tested the ability of the AptaNPs to measure the adenine-nucleotide content in yeast cells. Our results successfully demonstrate the potential for monitoring any metabolite of interest in living cells by selecting specific aptamers and embedding them in nanoparticles.

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

“…ATP (32). The nanosensor is based on an aptamer recognition element which has been turned into a signaling molecule using an aptamer switch probe (ASP) design (16).…”

“…Unfortunately the nanobiosensor reported in Ref. 32 was not specific for ATP and hence it was only possible to determine the total intracellular concentration of adenine nucleotides…”

“…nucleases in the cellular environment. The ASP used is a molecular beacon-type probe containing the aptamer sequence and a complementary hairpin-forming sequence separated by a polyethylene glycol linker (29,32). A fluorophore (Alexa Fluor 488) and a quencher (Black Hole 1) are covalently attached to the two ends of the sequence.…”

“…The even distribution of nanosensors confirms a cytoplasmic location. In our previous report (32) we have excluded that the ASP is released from the nanoparticles and broken down by nucleases in the cytoplasm by the fact that insertion of control nanoparticles with a similar switch probe containing an unresponsive nucleotide sequence instead of the aptamer showed mainly fluorescence from the reference dye. Furthermore, it was confirmed that the switch probe in the control nanoparticle is functional, i.e.…”

Aptamer-based molecular imaging

“…Binding of ATP to the ATP aptamer in the probe changed the hairpin structure, resulting in Texas red moving away from Black Hole 2 and therefore increasing fluorescence. The probe was applied to image and measure total intracellular ATP concentration (2.9-5.1 mmol/L) (Nielsen et al, 2010). Using the same design but with a newly selected ATP aptamer that can distinguish ATP from ADP and AMP and the reporter-quencher pairs of Alexa Fluor 488-Black Hole 1 and Texas red-Black Hole 2, the same group developed new probes to image and measure the dynamics of intracellular ATP in yeast with time resolution of seconds and ATP concentrations of 0.5-8 mmol/L.…”