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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
Fig.3 The upper part of (1) and applied sections of calculation
Fig.4 The upper part of (2) and applied sections of calculation
Fig.5 The upper part of (3) and applied sections of calculation
Fig.6 The upper part of (4) and applied sections of calculation
About each RNA of (1)-(4), we calculate the sum of value of probability in applied sections mentioned above.
And we evaluate stability of structure in each mRNA when we set the value of (1) for 1.
【Result of calculation】
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 |
【Consideration】
The value of efficiency of circularization calculated in our experiment become like table3. (And please look at our RESULT page)
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 |
When we compare the value of modeling with that of experiment, their tendency is similar.
About phenomenon of Ⅱ
Splicing site in mRNA combine with each other and mRNA cyclize by nucleophilic substitution reaction. As described above, the nearer distance of splicing site, the stronger the force of two splicing sites pulling against each, so the cyclization will be easier.
Fig.7 Distance of both splicing sites in each mRNA
In this figure, the arrows show distance of splicing site in each mRNA.
We calculate the relativity distance of both splicing sites in each mRNA when we set the value of (1) for 1(significant figures are three columns.).
At the moment, we intend to adopt the value that took these numerical reciprocal numbers as the magnification.
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 |
【consideration】
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 |
When we compare the value of modeling with that of experiment, they are quite different.
Why did we get data like this? We consider what points were wrong in modeling when we regard experiment value as correct. The reasons which we can consider are following two contents.
As mentioned above, real shape of mRNA is tertiary structure. But we use RNA models of secondary structure as reference, because to predict RNA models of tertiary structure is too difficult for us. The RNA models of secondary structure and tertiary structure are quite different, so complementary sequences combining each other are different. Therefore the reliability of circularize efficiency of Ⅱ is quite low. And we consider that this is the worst point.
【Conclusion】
The value of phenomenonⅡdoesn’t correlate with the value calculated from the experiment. However, we may say that the value of phenomenonⅠ correlate with result the value calculated from the experiment.
From this result, stability of the hydrogen bonding of RNA can become one of the factor when we think of efficiency of splicing reaction by ribozyme.
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