Part:BBa_K4636007

Probe_0101802_bsj

Main

Probe_0101802_bsj 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_0101802) from hsa_circ_0101802. 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_0101802 .(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.

Criteria for designing probes

Binding sequence is mainly a feasible binding sites for cDNA of hsa_circ_0101802. We use blast to confirm that its binding 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.

Design a probe recognizing back splicing junction (BSJ)

In circRNA synthesis process, 5' and 3' ends of linear RNA precursors connects together, forming a specific back splicing junction (BSJ) site. Back splicing junction (BSJ) offers a higher degree of specificity for recognition. Thus, we designed probe to identify the cDNA of the back splicing junction (BSJ) of circRNA synthesized in our experiments, in order to elevate specificity for probe detection.(Refer to Figure 3 for a detailed illustration of the BSJ of circRNA.)

Figure 3. Back splicing junction of circRNA.

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