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Team:HokkaidoU Japan/Projects/H Stem
From 2014.igem.org
Overview
The weak point of silencing by anti-sense RNA (asRNA) is the instability of RNA. RNA has a shorter half-life than DNA, it is essential to be stabilized for asRNA to repress mRNA sufficiently. Nakashima N and his colleagues solved this problem. They inserted each-complementary sequeces called stem regions into upstream and downstream of as RNA. These regions make asRNA form stem-loop structure, which leads to increase the stability of asRNA dramatically.
We followed in their footsteps. We designed our unique stem sequence called H-stem and producecd a BioBricked vector for asRNA expression so that other iGEMers and researchers can easily use stemed asRNA. Using this vector. Using this vector, you can gain an asRNA-expressing construct just by inserting PCRed target gene into the vector.
asRNA stabilization by stem sequence
The stem regions flank asRNA region as shown in Fig. 1 on DNA. Transcripted to RNA, stem sequences bind each other and make the whole RNA form stem-loop structure like Fig. 2.
This stem-loop structure contributes to RNA stabilization on the following two points.
One is the protection from the exonuclease activity of RNase. Since RNase in the cell decomposes single-strand RNA, stemed asRNA is hardly affected by such a degradation.
The other is an increase of thermodynamical stability by a decrease of Gibbs energy. Stem region produces such a great amount of negative delta G that positive delta G produced by the curvature of loop region is overwhelmed.
In bacterial cells, the decomposability of asRNA to that of target mRNA affects the amount of translated mRNA as shown below. Here, x-axis means the expression level of asRNA to that of mRNA.
From that graph, you can see that the stability of asRNA greatly contriburtes to the repression of mRNA. For the detail, please refer to the following.
The reaction of asRNA (hereinafter referred to as X), mRNA (Y) and RNA duplex (Z) consisting of the asRNA and the mRNA in a bacterial cell is
\[ X+Y \overset{k_{\rm unbind}}{\underset{k_{\rm bind}}{\rightleftharpoons}} Z \]Besides, mRNA and asRNA are produced and decomposed at all times. So, the ordinary defferential equation that represets the whole reaction in the cell is shown below.
\begin{cases} \dot{x}=a-bx-k_{\rm bind}xy+k_{\rm unbind}z & \\ \dot{y}=1-y-k_{\rm bind}xy+k_{\rm unbind}z & \\ \dot{z}=k_{\rm bind}xy-k_{\rm unbind}z-cz & \end{cases}Here, we took 1 for two constants of the right side first and second terms in the second formula using the flexibilities of [time] and [concentration]. Thus, constant \a stands for the expression level of asRNA to that of target mRNA and constant \[ b \] the decomposability of asRNA to that of mRNA.
The unique stable fixed point (in quadrant I) of the ODE is easily found by taking 0 for each equation. Especially, \[ y \] value of the fixed point, \[ y* \], is
\[ y=\frac{1}{2} \biggl\{ \sqrt{ \bigl( a-1+\frac{b}{\gamma} \bigl)^2 +4 \frac{b}{\gamma}} - \Bigl( a-1+\frac{b}{\gamma} \Bigl) \biggl\} \]Provided that \[ \gamma = \frac{k_{\rm bind}c}{k_{\rm unbind}+c} \]
We find the graph above by regarding this formula as a function of $a$.
Though here we let \gamma value be 1 arbitarily, this value does not affect qualitatively as the formula shows.
