Real-time PCR detection of protein analytes with conformation-switching aptamers

Abstract

We have developed a novel method that utilizes conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte, and a different secondary structure in the presence of a protein such as thrombin or PDGF. The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (since no signal is produced by background binding) and to the proximity ligation assay (PLA; since only one epitope on a protein surface must be bound, rather than two).

Aptamer-based biosensors for biomedical diagnostics

“…6) Finally, 27 one strategy to address the sensitivity problem is to perform signal amplification. To achieve this goal, a large number of methodologies have already been employed, such as using metallic nanoparticles, 67 quantum dots, [171][172][173] DNA machines, 154,[174][175][176] PCR, 177 and rolling circle amplification (RCA). 178 An interesting concept, called binding induced self-assembly, takes advantage of proximity or increased local concentration.…”

Enzyme-free dual amplification strategy for protein assay by coupling toehold-mediated DNA strand displacement reaction with hybridization chain reaction

“…Nevertheless, since the level of many proteins such as hormone, growth factor, etc., are ultra-low, sensitivity of many aptasensors is still unsatisfactory. On the other hand, although some signal amplification approaches have been introduced to improve the sensitivity of protein detection [3], for instance, nucleotide polymerasebased strand displacement [4], rolling circle amplification (RCA) [5], polymerase chain reaction (PCR) [6], nicking endonuclease-based signal amplification [7], and Exo III-assisted signal amplification [8,9]. These enzyme-based amplification strategies usually require different protein enzymes, whose characteristics, including specific reaction conditions and reaction-time dependence, may restrict the utilization of these aptasensors.…”

Evaluation of aptamer specificity with or without primers using clinical samples for C-reactive protein by magnetic-assisted rapid aptamer selection

“…8,9,17,[21][22][23] The detailed procedures are described in the Experimental section. The tting curve is shown in Fig.…”

“…In this work, the K d value of selected aptamers was determined by q-PCR. 8,9,17,[21][22][23] The process is described briey as follows: a series of progressively diluted reacting aptamers reagents (1000 nM to 7.8125 nM) in 20 ml of BD buffer were heated to 95 C for 5 minutes and cooled at 4 C to form secondary structures. A partial series of diluted aptamers was retained for input control (input).…”