Team:OUC-China/Model

Team OUC-China: Main

Overview

The aim of our project is to develop a better post-transcriptional regulation strategy and use it in monocistron and polycistron. We build models to design and predict our work.

miniToe —— a better transcriptional regulate strategy

To achieve a better post-transcriptional regulation strategy, we design a system which is composed of an RNA endoribonuclease (Csy4) and an RNA module named miniToe. We model to describe the dynamics of the miniToe system and point out the way to achieve different regulation level. The ODE and molecular dynamics are two main tools to explore it. We use the ODE to describe the reaction curve and the molecular dynamics give some explanations to experimental data.

Below you can follow the several questions we point out to have a better understanding of model work and the miniToe system. We will discuss some structures of Csy4 in different stage (Q1), some structures of miniToe system in different stage (Q2), the reaction order and some keys of miniToe system (Q3), the simulation of ODE model (Q4), some significant symbol in molecular dynamics (Q5) and the way to different regulation level (Q6).

Q1 : What does the structure of Csy4?

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The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin which consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. The Fig.1 and Fig.2 shows two structure of Csy4: with and without hairpin bound.

Fig.1 The structure of Csy4 without hairpin bound (PDB ID: 4AL5, resolution 2.0 A)

Fig.2 The structure of Csy4 with hairpin bound (PDB ID: 4AL5, resolution 2.0 A)

Q2 : What does the structure of miniToe structure?

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Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4. The whole system works as a translational activator including three modular parts:

1. A cis-repressive RNA (crRNA) to serve as translation suppressor by pairing with RBS as the critical part of miniToe structure.
2. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
3. A CRISPR endoribonuclease Csy4.

Fig.2-1 is the secondary structure of miniToe.

Fig.2 The structure of miniToe.

The Fig.4 and Fig.5 show that the two complex of miniToe structure: with and without specific site of hairpin cleaved, which is called the precursor complex and precursor complex respectively.

Fig.4 The precursor complex of wild-type Csy4

Fig.5 The product complex of wild-type Csy4

Q3 : What is the reaction order and key points of miniToe system?

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Fig.6 The working process of miniToe system

All the reaction happened in our first system, miniToe, can be described chronologically by following five main steps in Fig.6:

(1)The miniToe structure is produced and accumulated.
(2)The Csy4 is produced with IPTG induced.
(3)The Csy4 binds to the miniToe structure and form the Csy4-miniToe complex
(4)The Csy4 cleave the special site and divide the miniToe structure into two parts: the Csy4-crRNA complex and the mRNA of sfGFP.
(5)The sfGFP is produced.

From the description above, we can get four key problems in our system to make sure that our system can work successfully:

(1)Does the Csy4 dock correctly with the miniToe structure (hairpin)?
(2)How about the ability of binding between the Csy4 and miniToe structure (hairpin)?
(3)How about the ability of cleavage between the Csy4 and miniToe structure (hairpin)?
(4)Does cis-repressive RNA release from the RBS?

Q4 : How about simulation result of the ODE model?

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According to the work process we build an ODEs model and simulate our miniToe system for 30h, the result can be seen in the Fig.7.

Fig.7 The dynamics of sfGFP by model prediction

We compare the experimental data to the simulation, find it fit perfectly in Fig.7

Fig.8 The comparison between experimental data and simulation data

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Q5 : How about simulation result of the molecular dynamics?

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The main work our molecular dynamics is to give an explanation for the experimental data at atom level and four key points we discuss before: the experiment have proved that the miniToe structure is working well which means the four key points is work well too. We will present you the result of molecular dynamics following the four key points.

For the first key point, we have the interaction matrix to describe the molecular docking, and the heatmap of matrix can be seen in Fig.9.

Fig.9 The heatmap of interaction matrix for wild-type Csy4.

For the second problem, we calculated the free binding energy of Csy4/RNA complex. The result of binding free energy for wild-type Csy4 is

△ G_{b i n d i n g} = - 59154.9251 \overset{}{} k j / mol

.

For the third key point, we check the distance of Ser151(OG)-G20(N2’), which is a key interaction in the active site of Csy4 to describe the ability of cleavage. The distance curve of Ser151(OG)-G20(N2’) for wild-type Csy4 can be seem in Fig.10.

Fig.10. The distance of Ser151(OG)-G20(N2’) in wild-type Csy4

For the last key points, we use the RMSD of product to describe the release of crRNA. The result can be seen in the Fig.11.

Fig.11. The distance of Ser151(OG)-G20(N2’) in wild-type Csy4

The RMSD is unstable which give an explain to experiment that crRNA is release from RBS.

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Q6: How to achieve the goal of different regulate level?

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When we make the sensitivity analysis for our ODEs model, we find that while the cleavage rate of Csy4 will influence the expression in GFP, which indicated if we change the wild-type Csy4 to some mutation then we can achieve the different expression of GFP. The Fig.12 is showing that what will happen in the GFP expression curve if we change the cleavage rate of Csy4. It can achieve the goal of different regulate level.

Fig.12 The curve of sfGFP with the changing cleavage rate

miniToe Family —— The way to fifferent regulate level

In the miniToe family, the protein and hairpin are mutated to meet the goal of the different regulation level. In this part, the model helps to design mutants. For the own feature of Csy4 and hairpin, different strategies are used to design: molecular dynamics plays an important role in designing protein mutants while the bioinformatics and machine learning support us to choose hairpin mutants.

We are going to discuss the method to design Csy4 mutants (Q7)，how the method work in design and the result (Q8), the problem different from Csy4 designing when design the hairpin mutants and how to solve it (Q9) and the result of the mutants designing (10).

Q7: How to design the Csy4 mutants?

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The wet lab members give us four important sites, Gln104, Tyr176, Phe155, His29, which play important roles in binding and cleavage in protein Csy4 which can be seen in Fig.13. Considering 20 kinds of amino acids, we have 80 mutants to explore and choose if we only have one site mutated.

Fig.13 The four importatnt site in Csy4

In Q3, we point out four key points which will directly influence the work of our miniToe system. And in Q5, according to the molecular dynamics, we have four significant symbols to describe the four key points.

Now we are going to construct an logic line to show you how to use the three main information above to designing the Csy4 mutnats:

What we know and proved by the experiment is that the wild-type Csy4 with the miniToe system is working well, which means that all the important key points we discussion did not exist in the wild-type Csy4. The wild-type Csy4 can dock correctly with the miniToe structure and the Csy4 have a good ability to bind and cleave the miniToe structure, finally the crRNA release from the RBS. So we choose the wild-type Csy4 as a standard, and all the Csy4 mutant can check the four key points by comparing to wild-type Csy4.

Now for the four key points in Q3 we have something in mathetical forms to describe it in Q5. The most important thing is transform to the problem that how to make an comparison between mutant and wild-type Csy4 .

Q8: How does the design methods work?

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In Q7, we have the full logic lines in designing the Csy4 mutnats. Then here we will give the comparison method for the four key points in miniTOe system between mutants and Csy4 wild-type.

Now we have four mathematical forms ingcluding two curve, an numerical value and a matrix.Four things can divided into two kind of data: the matrix and the numerical value. The interaction matrix and the curve can be regard as matrix because the curve is discrete, and the binding free energy is just an numerical value.

For the matrix we can use Euclidean distance to describe the difference between two matric:

D (p, q_{W T}) = \sqrt{\sum_{i}^{m} \sum_{j}^{n} {(p_{i, j} - q^{W T}_{i, j})}^{2}}

For the free bind ing energy, we used the formula below to calculate the difference between the wild type and mutant[20]:

\ln (K_{d r e l}) = \ln (\frac{K_{d W T}}{K_{d M U T}}) = △ △ G_{b i n d i n g}

According to description above, we define four value used to compare four key points between mutant and wild-type:

D_{1} (int t e r a c t i o n \overset{}{} m a t r i x)

,

\ln (K_{d r e l})

,

D_{3} (S e r 151 - G 20 \overset{}{} c u r v e)

,

D_{4} (R M S D)

.

For the mutant Q104A, the four is showing in the following chart

Csy4-Mutant	$D_{1}$	$\ln (K_{d r e l})$	$D_{3}$	$D_{4}$
Q104A	0.483	2483	9.48	30.82