DNA

Part:BBa_K4029012

Designed by: JUAN LIANG-HSUEH   Group: iGEM21_CSMU_Taiwan   (2021-10-14)


Circular DNA corresponding to target miR-664a-3p

Introduction

BBa_K4029012 is a sequence for Circular DNA corresponding to target miR-664a-3p consisting of 75nt. It includes IMP binding site BBa_K4029002, miR-664a-3p binding site BBa_K4029003.

Function

The function of BBa_K4029012 is to serve as the template sequence for the RCA reaction by Phi29 polymerase, of which the reaction product is a series of DNA products consisting of repeated sequences of BBa_K4029012 used as indicators of the amount of our biomarker miR-664a-3p in the test sample.

Design

BBa_K4029012 was designed using the Circular DNA Design software (for more information, please refer to our model page https://2021.igem.org/Team:CSMU_Taiwan/Model). Through the python language, we derived this sequence of DNA of which we then ran through three DNA structure testing websites (RNAstructure, Kinefold, UNAFold) to verify the stability and secondary structure of this part, of which the results are shown below.

Model Prediction

UNAFold analysis result [1]


Figure 1. RNAStructure analysis result
Figure 2. KineFold analysis result


















Experiment Result

Circularisation of Linear DNA


Circular probe was synthesized by ligation of the linear DNA. 30 μL reaction solution containing 1 μM circle primer, 1 μM ligation primer, T4 DNA ligase buffer and 10U/ μL T4 DNA ligase was incubated at room temperature for 4.5 h. Then, the linear DNA that were not ligased and ligation primers were digested by adding Exonuclease I and Exonuclease III. Finally, denaturate the enzyme at 80 °C for 20 min.
We took circularized DNA to run gel-electrophoresis, evaluating the result of DNA ligation.

Fig.1

Circularized DNA were analyzed by agarose gel electrophoresis. M: marker, 1:T4 ligase primer, 2: linear DNA, 3: CPB-664a-3p, 4: CPB-33b-3p

The gel showed a “smear” in the lane of circular DNA probe after ligation, which is a common observation in circular DNA gel-electrophoresis due to its conformation.
However, we are aware that the "smear" cannot confirm successful circularisation of linear DNA, as there is a possibility of contamination as well.


RCA action and Linear DNA Circularisation


As we have only used our software model (for more information, please refer to Model) to design our circular probe sequence, in this segment of our experiment, we attempted to verify binding of target miRNA to our circular probe, as well as circularisation of our Circular Probe.

Fig.2

Verification of Circular probe 664a-3p. ***p < 0.001: Ctrl w/o miRNA compared to miRNA 664a-3p. ***p < 0.001: Ctrl w/o phi29 compared to miRNA 664a-3p. CBP: Circular Probe

Fig.3

Verification of Circular probe 33b-3p. ***p < 0.001: Ctrl w/o miRNA compared to miRNA 33b-3p. ***p < 0.001: Ctrl w/o phi29 compared to miRNA 33b-3p. CBP: Circular Probe

As seen in the above graph, it can be interpreted that our test with the RCA reaction mixture was successful in carrying out the desired mechanism.
Comparing the test with RCA reaction mixture to the control without phi29 polymerase in the mixture, it can be seen through the absorbance value that the presence of phi29 polymerase has greatly reduced DNA product output significantly.
From the above, we can establish the following:

  1. As the magnitudes of difference between the test with the RCA mixture and both controls are very significant, it can be concluded that RCA reaction was indeed carried out.
  2. For the RCA reaction to be carried out, it can be further concluded that the linear DNA sequence of our Circular DNA Probe was successfully circularised into a Circular DNA Probe and that the target miR has bound to our Circular DNA Probe to act as a primer to start RCA.

Phi29 polymerase action and Optimal Reaction Time


We have also conducted RCA experiments at different durations.

Fig.4

Fig.5

From the graphs above, it can be seen that there is an upwards trend from 0.5h to 2h. From the above information, we can deduce that there was indeed phi29 polymerase elongation reaction over time, eliminating the possibility of absorbance value from other factors other than phi29 action in RCA.
However, it is also to be noted that at the 3h mark, there is an observed decrease in absorbance compared to before. This suggests degradation of DNA product from RCA.
Therefore, we have decided to treat 2h as the ideal duration for our kit for highest efficiency.


RCA action against miR concentration


Finally, we attempted to validate the concept of our mechanism by testing different concentrations of target miRs to derive fluorescence intensity.

Fig.6

Fig.7

As seen above, it can be seen that there is an increase in fluorescence absorbance with increase in miR concentration in both cases.
From here, we can that given the above two verification tests, it can be said that an increase in target miR concentrations led to an increase in amount of product DNA.
We are well aware that our experiment result here is not sufficient enough of a set of data to completely verify the validity of a trend in our kit. However, due to the restrictions imposed by the covid-19 pandemic as well as time constraints, we were unable to do further tests on more concentrations of miR.



Sequence and Features


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]


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