C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP

  • Min S. Wang
  • Joshua C. Black
  • Michelle K. Knowles
  • Scott M. Reed
Original Paper
First Online:
Received:
Revised:
Accepted:

Abstract

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.

Figure

Evidence for mCRP selectivity of aptamer by gel electrophoresis, fluorescence anisotropy, and TIRF microscopy

Keywords

C-reactive protein Isoform Aptamer Cardiovascular disease TIRF microscopy Fluorescence anisotropy 

Abbreviations

CRP

C-reactive protein

CVD

Cardiovascular disease

TIRF

Total internal reflection fluorescence

RNA

Ribonucleic acid

DNA

Deoxyribonucleic acid

SELEX

Systemic evolution of ligands by exponential enrichment

NIR

Near infrared

DEPC

Diethylpyrocarbonate

EDTA

Ethylenediaminetetraacetic acid

ELISA

Enzyme-linked immunosorbant assay

SDS

Sodium dodecyl sulfate

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

DTT

Dithioreitol

PBS

Phosphate-buffered saline

SPR

Surface plasmon resonance

BSA

Bovine serum albumin

PAGE

Polyacrylamide gel electrophoresis

EMSA

Electrophoretic mobility shift assay

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Notes

Acknowledgements

Support from NIH 1R15GM088960-01 (SMR), NSF CBET-1033161 (SMR), and NSF CBET-1033215 (MKK) is acknowledged. We thank the UCD Biology department for use of the Odyssey imager.

Supplementary material

216_2011_5174_MOESM1_ESM.pdf (47 kb)
ESM 1 (PDF 46.7 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Min S. Wang
    • 1
  • Joshua C. Black
    • 2
  • Michelle K. Knowles
    • 2
  • Scott M. Reed
    • 1
  1. 1.Department of ChemistryUniversity of Colorado DenverDenverUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of DenverDenverUSA

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