Display options
Cite
Hu J, Kim J, Easley CJ. Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis. Anal Methods. 2015;7(17):7358-7362doi: 10.1039/c5ay00837a.
Hu, J., Kim, J., & Easley, C. J. (2015). Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis. Analytical methods : advancing methods and applications, 7(17), 7358-7362. https://doi.org/10.1039/c5ay00837a
Hu, Juan, et al. "Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis." Analytical methods : advancing methods and applications vol. 7,17 (2015): 7358-7362. doi: https://doi.org/10.1039/c5ay00837a
Hu J, Kim J, Easley CJ. Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis. Anal Methods. 2015 Sep 07;7(17):7358-7362. doi: 10.1039/c5ay00837a. PMID: 26366207; PMCID: PMC4562029.
Copy Download .nbib Format: NLM
Share it on

Anal Methods. 2015 Sep 07;7(17):7358-7362. doi: 10.1039/c5ay00837a.

Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

Analytical methods : advancing methods and applications

Juan Hu, Joonyul Kim, Christopher J Easley

Affiliations

  1. Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, AL 36849.

PMID: 26366207 PMCID: PMC4562029 DOI: 10.1039/c5ay00837a

Abstract

Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (-dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument-even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding.

References

  1. Electroanalysis. 2009 Jun 1;21(11):1267-1271 - PubMed
  2. Analyst. 2011 Sep 7;136(17):3461-8 - PubMed
  3. Anal Chem. 2008 Jul 1;80(13):5152-9 - PubMed
  4. Anal Bioanal Chem. 2006 Mar;384(5):1175-80 - PubMed
  5. Biochemistry. 1996 Nov 12;35(45):14413-24 - PubMed
  6. Angew Chem Int Ed Engl. 2013 Oct 4;52(41):10698-705 - PubMed
  7. Trends Biotechnol. 2011 Jan;29(1):1-5 - PubMed
  8. Analyst. 2011 Jan 21;136(2):257-74 - PubMed
  9. Nucleic Acids Res. 2011 Aug;39(15):e102 - PubMed
  10. Sci Rep. 2014 Mar 12;4:4360 - PubMed
  11. Nat Biotechnol. 2002 May;20(5):473-7 - PubMed
  12. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5426-30 - PubMed
  13. Electrophoresis. 2010 May;31(10):1615-22 - PubMed
  14. J Am Chem Soc. 2014 Jun 11;136(23):8467-74 - PubMed
  15. Angew Chem Int Ed Engl. 2010;49(2):355-8 - PubMed
  16. Biosens Bioelectron. 2014 Aug 15;58:308-13 - PubMed
  17. Anal Chem. 2015 Jan 6;87(1):821-8 - PubMed
  18. Clin Chem. 1993 Sep;39(9):1953-9 - PubMed
  19. Nucleic Acids Res. 2005 Apr 07;33(6):e64 - PubMed
  20. J Am Chem Soc. 2012 Apr 25;134(16):7066-72 - PubMed
  21. Chem Commun (Camb). 2013 Jul 14;49(55):6137-9 - PubMed
  22. Chembiochem. 2005 Dec;6(12):2163-6 - PubMed
  23. Nat Protoc. 2007;2(9):2212-21 - PubMed
  24. Physiol Rev. 1999 Oct;79(4):1283-316 - PubMed
  25. Anal Chem. 2010 Aug 15;82(16):6976-82 - PubMed
  26. Biosens Bioelectron. 2011 Mar 15;26(7):3346-52 - PubMed
  27. Anal Chem. 2011 Dec 1;83(23):8867-70 - PubMed
  28. J Biomol Screen. 2001 Dec;6(6):429-40 - PubMed

Publication Types

Grant support