Part:BBa_K4636010:Design

Probe_0004771_bsj

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]

Design

The sequence we designed can be separated into two parts: The binding sequence and the extension sequence. (See Figure 1 for details of probe sequence.)

Figure 1. 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_0004771. 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 2 for a detailed illustration of the BSJ of circRNA.)

Figure 2. Back splicing junction of circRNA.

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