Real-time PCR detection of protein analytes with conformation-switching aptamers
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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2015, Analytica Chimica ActaCitation Excerpt :For example, Chen and co-workers have selected DNA aptamers targeting AMACR by single-bead SELEX, which can be further applied to the fluorescent detection of AMACR [6]. In recent years, many highly sensitive aptamer-based assays have been proposed for the detection of protein via different enzymes based signal amplification [21–24]. Although exhibiting high sensitivity, these assays require complicated experimental design or operation.