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

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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.

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

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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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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.

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Authors and Affiliations

  1. Department of Chemistry, University of Colorado Denver, Denver, CO, 80217-3364, USA

    Min S. Wang & Scott M. Reed

  2. Department of Chemistry and Biochemistry, University of Denver, Denver, CO, 80208, USA

    Joshua C. Black & Michelle K. Knowles

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  1. Min S. Wang
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Correspondence to Scott M. Reed.

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Wang, M.S., Black, J.C., Knowles, M.K. et al. C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP. Anal Bioanal Chem 401, 1309–1318 (2011). https://doi.org/10.1007/s00216-011-5174-1

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