NUPACK Guide
In collaboration with Team Aboa, we created this guide to lower the barrier of entry to these kinds of programs for life science students. Computational calculations and simulations are of growing importance in synthetic biology and life sciences in general, yet they can be quite intimidating for beginners. This is not meant to be the most comprehensive tool for creating complex programs but rather an easily digestible compilation of information for beginners and support during their first endeavors in programming. We hope that this guide encourages more people to utilize computational tools in general and therefore helps build the skill-set of life science researchers. Although this is not aimed at experienced users, they can still find this helpful, as we also learned more about this program by writing this guide.
APTAMER BOOK: An Introduction to Aptamers
In collaboration with Linköping, NYU Abu Dhabi, Nazarbayev University, IISER Mohali, we published this Aptamer book, for future researchers and iGEMers have an easy-access to tons of relevant information for Aptamers. This book consists of brief introduction to aptamers, a guide to MAWS, tons of applications of aptamer, compilations of previous iGEM projects and a lot more.
GUIDEBOOK: Molecular Dynamics Simulation of RNA-Ligand Complex
Tackling with heavy codes and high performance computers to produce any in-silico molecular dynamics simulations can be a scary task and to learn the language of the machine is extremely difficult. So to help the beginners achieve these MD simulations, we tried to compile a simple solution.
This guide provides step-by-step information specifically for RNA-Ligand molecular dynamics and is a self tested guide as we used it to achieve the contribution in Spinach 2.1 aptamer flanked by tRNALys3(Part:BBa_K1330000). The guide was compiled by our team and the MD simulations were assisted by Mr Arijit Saha, an external collaborator.
ARDUINO CODE: For fluorescence detection using photodiode
For our project, we used photodiode, arduino board and LCDs to visualise the the fluorescence intensity from a biological fluorescence.
Description of the code: In our system we have a photodiode connected to the Arduino. The arduino board takes an analog signal input from the photodiode circuit and gives an output in the LCD screen. As the intensity of the light increases,the current output from the photodiode increases.The Analog signal undergoes conversion and is compared with a threshold value. The LCD screen displays High when the value if is greater than a threshold value and LOW otherwise. The future iGEM teams who wants to configure Arduino code to run a similar system can use this code.
To read more about this, go to Hardware.
Click here to access the code as a PDF.
STANDARDISED PROTOCOL FOR SELEX
Aptamers are generated by an in vitro evolution method known as the Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX experiments can be conducted against various target molecules or elements, such as small compounds, proteins, nanoparticles, or live cells. As a contribution to future iGEM teams, we would like to contribute the protocols for synthesising DNA library and purification and desalting protocols. These protocols have been standardised and have been tested. See Results for more.
CONTRIBUTION TO BBa_K1330000:
A. Potential Spinach 2.1 mutants using Molecular Dynamic Simulations and MFE calculations with higher fluorescence intensity as compared to BBa_K1330000
Towards the modification of parts from the Registry of Standard Biological Parts (RSBP), we aimed to design investigations that would enhance the fluorescence intensity of Spinach 2.1(BBa_K1330000), a light-up aptamer. In order to achieve this, we altered the sequence by introducing mutations (single, double), inversions, and deletions at different regions of the Spinach2.1 aptamer sequence to increase its relative fluorescence intensity than the wild-type.
All possible single-point mutations (SPMs) were made in the tetraloop of Spinach 2.1 using combinatorics and inversions. The free energy of the thermodynamic ensembles, frequency of the minimum free energy (MFE) structure, and the ensemble diversity were predicted using RNAfold. Based on these predictions, we chose a few sequences that displayed the best parameters for the above predictions. The mutated sequences and their corresponding RNAfold structures are given Figure 1.
Mutations and Inversion:
The free energy of the thermodynamic ensemble are predicted to be -34.44 kcal/mol, -34.44 kcal/mol, -34.80 kcal/mol and -35.50 kcal/mol for the sequences (modified Spinach2.1) of Spinach2.2(BBa_K4438200), Spinach2.3(BBa_K4438201), Spinach2.4(BBa_K4438202) and Spinach2.5(BBa_K4438203) respectively.
Figure 1.Sequence and structures of the RNA Aptamers. The DNA sequence and the RNAfold structures of Spinach2 and Spinach2.1 are adopted from the part (BBa_K1330000), made by iGEM DTU Denmark. The Spinach2 sequences highlighted in bold are the aptamer sequence, and those flanking are the tRNA scaffold sequence, tRNALys3. The yellow colored box highlights the tetraloop sequence (colored red) and the bases adjacent (colored blue) to the tetraloop. The bases colored green are mutated from A/T and T/A to give a modified Spinach2 version, Spinach2.1. The other Spinach aptamer versions are derived from the Spinach2.1 sequence by base mutations in the tetraloop and the region adjacent to it (highlighted in the yellow colored boxes) and keeping the rest of the sequence unchanged. These mutants correspond to Spinach2.2, Spinach2.3, Spinach2.4 and Spinach2.5. The mutations in the DNA sequence are displayed on the RNAfold structures of the RNA aptamers (Spinach). The tetraloop region is indicated by a box colored blue for Spinach2 and the other mutations are circled in blue for other modified Spinach aptamers.
Using these sequences we ran GROMACS simulation to find the energy minimization results. These results were plotted using the steepest descent algorithm and it was observed that the native structure converged in almost 200 steps (Figure 2a) while for Spinach2.6 it converged in nearly 180 steps (Figure 2d). An NVT equilibration was performed and trajectory files were generated to visualise the simulation. It was found that the system reached equilibrium at less than 20 ps (Figure 4b, e). This equilibrated system was subjected to the Steer MD simulations to dislocate the ligand from the G-quadruplex binding site with spring force 10000 KJ/mol/nm2, see the Figure 2c, d. The force applied was able to pull the ligand apart from the binding site of the RNA aptamer as seen in the animation below. Overall, the energy minimization plot suggests that the Spinach2.6 (BBa_K4438204) complex structure is relatively stable when compared with the native structure. Moreover, the Steer MD plots for both these complexes are significantly different suggesting the binding affinities of the ligand to RNA aptamer structure are different. However, additional simulations and experimental confirmations need to performed to strengthen this observation
Overall, our in-vitro and in-silico results indicate that Spinach2.6 potentially show higher fluorescence intensity compared to native form.
Figure 2. Results of MD simulations of native and modified RNA aptamers:a, b) Energy minimization and equilibration plots of native (4TS2) complexes respectively. c) Steer MD simulation plot for the native structure. d, e) Energy minimization and equilibration plots of modified Spinach RNA aptamer, Spinach2.6 complex structure respectively and f) Steer MD simulation plot for the Spinach2.6 complex structure.
