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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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A turn-on fluorescent PCNA sensor.

Aimee J Horsfall1, Theresa Chav1, John B Bruning2

  • 1ARC Centre of Excellence for Nanoscale BioPhotonics, Institute of Photonics and Advanced Sensing, School of Physical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.

Bioorganic & Medicinal Chemistry Letters
|April 11, 2021
PubMed
Summary
This summary is machine-generated.

New fluorescent sensors were developed using modified peptides to detect the proliferating cell nuclear antigen (PCNA). Peptide 3a shows promise for creating a PCNA-selective sensor for cancer research.

Keywords:
FluorescencePeptideProliferating Cell Nuclear Antigen (PCNA)SensorSolvatochromic amino-acid

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Proliferating cell nuclear antigen (PCNA) is a crucial biomarker for cell proliferation and is often dysregulated in diseases like cancer.
  • Developing sensitive and selective fluorescent sensors for PCNA is essential for disease diagnostics and research.

Purpose of the Study:

  • To engineer novel peptides incorporating solvatochromic amino acids (4-DMNA and 4-DAPA) as turn-on fluorescent sensors for PCNA.
  • To evaluate the sensing capabilities and binding affinities of these modified peptides with PCNA.

Main Methods:

  • Synthesis of p21 peptides (141-155) with 4-DMNA or 4-DAPA at specific positions.
  • Solvatochromic fluorescence measurements in polar and hydrophobic solvents.
  • Surface Plasmon Resonance (SPR) analysis to determine binding kinetics (KD) with PCNA.
  • Fluorescence-based titration assays with varying PCNA concentrations.

Main Results:

  • Peptides containing 4-DAPA exhibited a significantly higher fluorescence enhancement (300-fold) compared to 4-DMNA (40-fold) between different solvent polarities.
  • SPR analysis confirmed specific interaction of peptides 3a and 3b with PCNA, with dissociation constants (KD) in the nanomolar to low micromolar range.
  • Peptide 3a demonstrated a 10-fold fluorescence change upon binding to 2.5 equivalents of PCNA, indicating a sensitive turn-on response.

Conclusions:

  • Peptide 3a, functionalized with 4-DMNA at position 151, is a promising lead for developing a selective turn-on fluorescent sensor for PCNA.
  • This sensor has potential applications in monitoring cell proliferation for diseases such as cancer.