Part:BBa_K4636012

Probe_0004771_12

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Main

Probe_0004771_12 is a single-stranded DNA (ssDNA) that can conjugate to RCA product (It’s a kind of tandem repeat DNA of cDNA of hsa_circ_0004771) from hsa_circ_0004771 that we synthesized for method testing. In our experiments, samples with successful circularization can be recognized by this probe, and then provide for further detection through gold nanoparticle-based colorimetric assay.

In gold nanoparticle-based colorimetric assay, we will use probe-conjugated gold nanoparticles (Gold nanoparticles and ssDNA probe) complex to identify the amplification products of RCA. Further AuNPs-probe solution color changes after adding salt can be used to verify whether the sample contains a higher amount of hsa_circ_0004771 .(See Figure 1 for details of gold nanoparticle-based colorimetric assay)

Figure 1. Schematic diagram of gold nanoparticle-based colorimetric assay.

Design

The sequence we designed can be separated into two parts: The binding sequence and the extension sequence. (See Figure 2 for details of probe sequence.)

Figure 2. Probe design compose of binding sequence and extension sequence.

Binding sequence is mainly a feasible binding sites for cDNA of hsa_circ_0004771. We use blast to confirm that its specificity is high enough. In addition, we also have the following considerations:
(1) GC content is between 40%~60%.
(2) Tm value is around 40°C.
(3) Finally, we refer to the paper[1] and add two AA sequences as extension sequences at the 3’ end of the sequence. AA sequence is between the binding sequence and gold nanoparticle.
(4) The avoidance of self-dimer is also important for primer design.

Experiment

Gold nanoparticle and probe successfully joined :
Through the detection of nanodrop, we can find that the peak wavelength of AuNPs-probe has a slight red shift compared with gold nanoparticle without probe. According to the reference paper[2], this red shift can indicate that the probe has been successfully connected to the gold nanoparticle.

Reference

1. Ali, M. M., Kanda, P., Aguirre, S. D., & Li, Y. (2011). Modulation of DNA-modified gold-nanoparticle stability in salt with concatemeric single-stranded DNAs for colorimetric bioassay development. Chemistry (Weinheim an der Bergstrasse, Germany), 17(7), 2052–2056. https://doi.org/10.1002/chem.201002677
2. Li, F., Zhang, H., Dever, B., Li, X. F., & Le, X. C. (2013). Thermal stability of DNA functionalized gold nanoparticles. Bioconjugate chemistry, 24(11), 1790–1797. https://doi.org/10.1021/bc300687z