Part:BBa_K4636000

Modified T7 promoter

In our project, we have designed a T7 promoter upstream of the target sequences (cDNA of hsa_circ_0101802 and hsa_circ_0004771) for in vitro transcription. The T7 promoter sequence is provided below (see Figure 1). This promoter facilitates the binding of T7 polymerase for transcription, with transcription starting from the underlined point (Figure 1).

Figure 1. T7 promoter sequence.

Additionally, we have incorporated additional sequences both upstream and downstream of the T7 promoter to enhance its transcription capability. According to the reference papers [1] [2], our design has the potential for high-efficiency transcription. (Figure 2) In theory, our modified promoter can be added to most types of DNA for in vitro transcription.

Design

Figure 2. Modified T7 promoter.

Refer to previous study[1], [2], we add four parts of sequence to enhance the transcription ability :
(1) Upstream of the T7 promoter, from -18 to -21, there is a high AT-rich region. This enhances the binding affinity of the T7 polymerase for the DNA template.
(2) Upstream of the T7 promoter, from -22 to -27, there is a highly GC-rich region. This enhances the binding affinity of the T7 polymerase for the DNA template.
(3) Downstream of T7 promoter from +1 to +3 : With triple G (GGG) sequence. It can increase binding affinity of the T7 polymerase for the DNA template. (Transcription start from the first G)
(4) Downstream of T7 promoter from +4 to +8 : With highly AT rich. It can assist DNA template to form a initiation bubble which increases the initially transcription ability.

Experiment

Since the remaining DNA templates have been degraded by treating DNase I, the presence of a band at Lane 2 indicated a successful in vitro transcription (IVT) with the assistance of Modified_T7 promoter. (Figure 3)Please refer to Results-CircRNA synthesis for more details.

Figure 3. Lane 1: low range RNA ladder, Lane 2: Linear form RNA, Lane 3: Circularization product, Lane 4: Circularization product + RNase R, Lane 5: Circularization product + RNase R

Reference

1. Tang, G. Q., Bandwar, R. P., & Patel, S. S. (2005). Extended upstream A-T sequence increases T7 promoter strength. *The Journal of biological chemistry*, *280*(49), 40707–40713. https://doi.org/10.1074/jbc.M508013200
2. Conrad, T., Plumbom, I., Alcobendas, M., Vidal, R., & Sauer, S. (2020). Maximizing transcription of nucleic acids with efficient T7 promoters. *Communications biology*, *3*(1), 439. https://doi.org/10.1038/s42003-020-01167-x

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]