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PROJECT
MODELING
Efficiency of circularization of mRNA is proportion to probability of meeting 3’ and 5’ side of the intron which is in the both side of mRNA. In this study, we designed complementary sequences near the 5’side of the intron and the 3’side of the intron to improve the efficiency of circularization of mRNA.
If there are complementary sequences, both splicing sites approach and the reaction of circularization is easy to happen because these sequences make hydrogen bands.
Therefore we predict how much efficiency of circularization improved.
The generators used in this study are (1)
[BBa_K1332011]
(2)
[BBa_K1859026]
(3)
[BBa_K1859024]
(4)
[BBa_K1859025]
mRNA transcribed from these generators are the sequences of the following parts.
(1) the 3´side of the intron
[BBa_K1859008]
, RBS
[BBa_B0034]
, RFP
[BBa_K1332002]
, the 5´side of the intron
[BBa_1332009]
(2) the 3´side of the intron
[BBa_K1859015]
, RBS
[BBa_B0034]
, RFP
[BBa_K1332002]
, the 5´side of the intron
[BBa_K1859016]
(3) the 3´side of the intron
[BBa_K1859003]
, RBS
[BBa_B0034]
, RFP
[BBa_K1332002]
, the 5´side of the intron
[BBa_K1859004]
(4) the 3´side of the intron
[BBa_K1859001]
, RBS
[BBa_B0034]
, RFP
[BBa_K1332002]
, the 5´side of the intron
[BBa_K1859006]
(1)It is the sequence of expressing circular mRNA in last year’s study
(2)It has complementary sequences outside the splicing sites.
(3)It has complementary sequences inside the splicing sites.
(4)It has also complementary sequences inside the splicing sites.
(3) and (4) are the reverse of the position of complementary sequences.
(Please look at the experiment page)
Fig.1 prediction of secondary structure of 4 kinds of mRNA
In figure, black lines show 3’intron, 5’intronM and splicing site.
First, when we consider the circularize efficiency, we have following three assumptions.
We assume that only the shapes that are illustrated in following fig.1 exist in mRNA which is not circularized yet. In the other words, we think that circularization doesn’t happen in other structures which are supposed to exist.
We assume that circular mRNA are only monomers so we assume that dimer and the others are not formed. Therefore we think that circular mRNA is formed by reacting both splicing sites of one molecule of mRNA.
Real shape of mRNA is tertiary structure but we consider efficiency of circularization regarding it secondary structure. Because it is too difficult to model basing tertiary structure.
【Method of calculation】
We make following two assumptions, and calculate the efficiency of circularization in each phenomenon.
Ⅰ.In structure of mRNA indicated in fig.1, we assume that stability of all combinations between 3’ and 5’ side of the intron is a correlation with their possibility (possibility of being close to each other). Therefore we assume that stability of these combinations is a correlation with possibility of circularization.
Ⅱ.We assume that distance of both splicing sites in structure of RNA indicated in fig.1 is a correlation with possibility of circularization.
About phenomenon of Ⅰ
Each base’s colors of fig.1 indicate probability of binding base pairs complementary (we call it probability) and we express it in figure of 0~1. In this time, we calculate value of Ⅰ by using this parameter. Concretely, we divide color into 66 equal and assign the probability value to these colors (table1). And we make color (stability) of all combinations between 3’ and 5’ side of the intron correspond to the value and evaluate in system of addition.
nbsp; The range of sequence applying possibility of all combinations between 3’ and 5’ side of the intron is these following sections in devices.
BBa_1332003 and BBa_1332005 / BBa_1859015 and BBa_1859016 / BBa_1859001 and BBa_1859003 (Please look at fig.3-6)
Fig.2 the color using in Fug.1
Table1. Probability value of each color
Table 2. Efficiency of circularizationⅠin each mRNA
| (1) | (2) | (3) | (4) |
| The sum of probability value | 23.7 | 35.1 | 34.3 | 31.8 |
| Efficiency of circularizationⅠ(relativity value) | 1.00 | 1.48 | 1.45 | 1.34 |
Table 3. Efficiency of circularization in experiment value
| (1) | (2) | (3) | (4) |
| Efficiency of circularization (relativity value) | 1.00 | 2.05 | 1.22 | 1.34 |
Table 4. Efficiency of circularization in each mRNA
| (1) | (2) | (3) | (4) |
| The distance (relativity value) | 1.000 | 0.998 | 0.278 | 0.493 |
| Efficiency of circularization Ⅱ(relativity value) | 1.00 | 1.00 | 3.60 | 2.03 |
Table 5. Efficiency of circularization in experiment value (the same one mentioned above)
| (1) | (2) | (3) | (4) |
| Efficiency of circularization (relativity value) | 1.00 | 2.05 | 1.22 | 1.34 |
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