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Fluorescent labeling of Taqman oligonucleotide probes via Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry

Abstract

We describe an approach to the synthesis of TaqMan oligonucleotide probes that is based on Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry when oligonucleotides containing an internal alkynyl group at the pyrimidine position are labeled post-synthetically with a f luorescent azide. TaqMan probes were constructed with f luorescein in different internal positions and a BHQ1 quencher on the 3′-end. Our previously designed alkynylated deoxyuridine or deoxycytidine phosphoramidites have been employed for the synthesis of alkynyl oligonucleotides. It was demonstrated that the synthesized TaqMan probes can detect accumulation of PCR product in real-time. The closer to the label the 3′-terminal quencher, the higher the quenching efficiency, but the efficiency of probe hybridization to DNA template is reduced in this case.

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Abbreviations

BHQ-1:

Black Hole Quencher 1

CuAAC:

Cu(I)-catalyzed dipolar alkyne-azide [3+2]-cycloaddition

CPG:

controlled pore glass

DMTr:

4,4′-dimethoxytrityl

DIPEA:

N,N-diisopropylethylamine

Flu:

5-acylaminofluorescein residue

HBTU:

hexaf luorophosphate of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

MALDI-TOF:

matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

TaqMan:

linear oligonucleotide probes

ТВТА:

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine

ТЕАА:

triethylammonium acetate

PCR:

polymerase chain reaction

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Correspondence to S. V. Vasilyeva.

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Original Russian Text © S.V. Vasilyeva, E.A. Burakova, L.G. Zhdanova, M.S. Anisimenko, D.A. Stetsenko, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 1, pp. 51–58.

The paper is based on the materials of the “Chemical Biology 2016” conference; Novosibirsk, Russia, July 24–29, 2016.

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Vasilyeva, S.V., Burakova, E.A., Zhdanova, L.G. et al. Fluorescent labeling of Taqman oligonucleotide probes via Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry. Russ J Bioorg Chem 43, 43–49 (2017). https://doi.org/10.1134/S1068162017010113

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  • DOI: https://doi.org/10.1134/S1068162017010113

Keywords

  • real-time PCR amplification
  • fluorescent probe
  • phosphoramidite