Difference between revisions of "Part:BBa K3154001"

Latest revision as of 01:07, 22 October 2019

Second Generation Toehold Switch for hsa-miR-21-5p with GFPmut3b

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.

Sensor characterization was designed with toehold switch for hsa-miR-21-5p as the sensing element, miR21:antimiR21 complex as the analyte and the fluorescence of GFP reported as the sensor output. The working of the sensor follows the basic principle of second-generation toehold switches wherein upon binding of miR:antimiR complex, the toehold switch unfolds to its linear conformation exposing the ribosome binding site. This triggers the translation of reporter gene (GFP) following which its fluorescence could be measured. Thus, the concentration of analyte (miR21:antimiR21 complex) is expected to follow a linear relation with the GFP fluorescence intensity. The fluorescent plate reader (Biotek Synergy H1 Multimode Reader) was calibrated using sodium fluorescein with varying concentration and the linearity plot was constructed with R2 = 0.99. The fluorescence was measured with excitation at 395 nm and emission at 520 nm with the instrument gain set at 100. We were quite intrigued initially on the obtained fluorescence values for excitation of GFP at 395 nm, because this excitation wavelength is a characteristic of the wt GFP while we had used a mut3b version of GFP which has its characteristic excitation wavelength at 485 nm. So, what we observe here is a blue shift of the absorption spectra, which could possibly be due to anything in the PURExpress Protein Synthesis kit that is interfering in the fluorescence phenomenon. We carried out a control study to validate this and we obtained fluorescence value of ~400 units for the kit solution alone. In addition, the wt and mut3b variants of the GFP vary from one another by 3 mutations in the sequence leading to a protonated & deprotonated variations of the chromophore. The ratio of the protonated and deprotonated chromophore species in the sample decides the excitation wavelength of the GFP, plausibly from our understanding. Hence, in our case, we obtained the maximum fluorescence intensity at 385 nm excitation with 520 nm being the emission wavelength, possibly due to protonation effects in the reaction system. This was fixed as the instrument parameters for the specificity and concentration study. These instrumental parameters were set for all the following studies. The NEB (#E6800) kit was tested using the DHFR control provided by the manufacturer. The protein expression was confirmed by SDS PAGE using a standard protein ladder for 20 kDa. Specificity of toehold for miR-21-5p: In order to comment on the specificity of the proposed toehold switch, non-specific analytes such as miR-200a-3p and miR-29a-3p with a concentration of 100 nM each, were used as sources of interfering analytes (Figure 1). The readings at 4 h were taken into consideration as 2 h reading had less significant intensity and hence the trend could not be inferred. The values were normalized with the control wherein miR-200-3p showed a four-fold decrease in intensity while the miR-29a-3p had effectively ‘zero’ intensity in comparison to that of miR-21-5p. The minimal leakiness towards miR-200a-3p might be attributed to its sequence similarity with miR-21-5p in the region which binds to the trigger domain of the toehold switch. Thus, the toehold for miR-21-5p was found to be highly unobtrusive for miR-200-3p and miR-29a-3p implicating its specificity.

Specificity study of toehold for miR-21-5p

Concentration study of toehold for miR-21-5p: The concentration study was performed for 3-fold range of concentration (100 pM, 1 nM, 10 nM and 100 nM). The readings taken at 4 h were considered as the 2 h readings had less significant intensity for lower concentrations and hence the trend was not discernable. The graph illustrates an increasing trend from 100 pM to 10 nM and the intensity was found to saturate at 100 nM (Figure 2). This implicates the linear range to be between 100 pM and 10 nM. The limit of detection (LOD) was found to be 100 pM implying its responsiveness even at very low concentration.

Concentration study of the toehold switch for miR-21-5p after after 4 h

A calibration curve was plotted by taking log of concentration of miR21:antimiR21 complex normalized with 10 pM (Figure 3). The sigmoidal curve was seen to saturate at about 10 nM validating the linear range (R2 = 0.99). A heat map was also constructed to better substantiate the saturation and the expected linear range was corroborated.

Calibration curve for the characterized toehold switch for hsa-miR-21-5p

Sequence and Features