Elsevier

Analytical Biochemistry

Volume 380, Issue 2, 15 September 2008, Pages 164-173
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2008.05.018Get rights and content

Abstract

We have developed a novel method that uses 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 platelet-derived growth factor (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 (because no signal is produced by background binding) and the proximity ligation assay (PLA) (because only one epitope, rather than two epitopes, on a protein surface must be bound).

Section snippets

Materials

The sequences of the conformation-switching aptamers for the detection of thrombin were as shown in Fig. 3, Fig. 4 as well as in Fig. S1 in the supplementary material. The substrate for ligation was t.5′P (5′ p-GGTTGGTAGTCTCGAATTGCTCTCT), where 5′ p denotes a 5′ phosphate. Primers for PCR were t.F1 (5′-TGTGGTTGGTGTGGTTGGTT), t.F2 (5′-GGTTGGTTCATGGTCATATTGGT), t.R1(5′-GAGAGCAATTCGAGACTACCAACC), and t.R2 (5′-AGAGAGCAATTCGAGACTACC). All oligonucleotides except minor groove binding (MGB) probes

Adapting conformation-switching aptamers to PCR

Despite the demonstrated advantages of real-time PCR for quantitation, the real-time PCR detection of protein analytes is not routinely practiced. Most schemes for immunoPCR do not directly couple protein-binding amplification to PCR amplification, instead requiring wash steps and other processing prior to amplification. However, conformation-switching aptamers can be used to transduce analyte-binding signals into optical and other signals [19], [20], [21], [24], [25], [26]. Therefore, rather

Conclusions

Although conformation-switching aptamers have been adapted to the detection of protein analytes in a variety of formats, there is an inherent difficulty in this approach. To have low background and a high signal/noise ratio, it is essential that the nonbinding conformation of the aptamer be much more stable than the binding conformation. However, the more stable the nonbinding conformation of the aptamer is, the more binding energy will be required to stabilize the binding conformation and the

Acknowledgment

We acknowledge the National Institute of Biomedical Imaging and Bioengineering (NIBIB) and the Welch Foundation for support.

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