Part:BBa_K3154000

Second Generation Toehold Switch for hsa-miR-21-5p

Toehold switches are a class of de novo designed mRNA based riboregulators that possess unique properties such as low crosstalk, high orthogonality and high dynamic range. They are hence highly desirable as agents of translational regulation of reporter protein genes in abiotic situations for many synthetic biology applications. Toehold switches have free energy and sequence constraints amongst the various domains that determine their optimal conformation. The regions of the toehold switch are the unique toehold or switch domain to which the trigger RNA binds via base pairing, the bottom stem, the secondary loop that contains the start codon, the top stem containing a scar site in the descending portion, the primary loop that consists of a preRBS and RBS sequence, and a horizontal linker sequence that concatenates the toehold switch to the downstream reporter gene. In the OFF state, the RBS cannot come in contact with the ribosome, and therefore no translation occurs. In the ON state, the unfolding of the switch into a linear molecule allows for the ribosome- RBS interaction to take place and translation to occur.

This part is a de novo engineered second-generation toehold switch for detecting the microRNA sequence hsa-miR-21-5p, wherein we recommend this part for its utility in the regulation of the downstream reporter gene followed by subsequent quantification of the microRNA hsa-miR-21-5p. The second-generation toehold switch design allows for higher programmability of the involved sequences, resulting in better performance of the toehold switch. The significance of the second generation design is necessitated by the presence of a stop codon in the open reading frame of the sequence of hsa-miR-21-5p. The toehold domain and the ascending segment of the bottom stem must be complementary to the trigger RNA in order to linearize the switch in its ON state. The descending segment of the bottom stem will thus be complementary to the ascending segment, resulting in a subsequence of the trigger itself. This allows for the stop codon to repeat itself in the reading frame after the start codon in the secondary loop. Therefore, translation stops at that point and no downstream gene can be expressed. The stop codon in the open reading frame of the switch sequence can be avoided by modifying the trigger sequence binding to it. An antisense RNA, or antimiR, is designed such that it is partially complementary to the microRNA. This creates a hybrid, T shaped trigger sequence where the base-paired stem of 12 nt length sequesters the stop codon, and the free ends of 22 nt in total, hybridize with the toehold switch.

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NUPACK image model of second generation toehold switch for hsa-miR-21-5p

Information contributed by City of London UK (2021)

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

• Group: City of London UK 2021
• Author: Lucas Ng
• Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

• Media:BBa_K3154000_thtest.txt
• Media:BBa_K3154000_crt.txt

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.

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This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37°C. We tested it against every mature Homo sapiens miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-6762-3p.

With hsa-miR-6762-3p at 37°C, the switch has a specificity of 42 ± 50 % and an activation of 20 ± 7 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here. CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:

Error bars represent the standard deviation. The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.

Caveats:

• As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.

We do not recommend this part for future usage.

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
  INCOMPATIBLE WITH RFC[1000]

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