Final Report Summary - MUDLOC (Multi-Dimensional Lab-On-Chip)
MuDLOC is a multi-dimensional microfluidic based platform for proteomics. The core technology of MuDLOC is a DNA to protein array. This means that a DNA array is turned into a protein array by in vitro transcription and translation inside the device, at nanoliter volumes.
The advantages of this approach are many. For example, there is no need to purify proteins in advance. The method is also compatible with all protein types including, as we demonstrate, thousands of membrane proteins. Protein functionality is a serious drawback of most protein arrays, we demonstrated that the fresh proteins produced on the MuDLOC platform are functional. This is a significant improvement over previous protein arrays.
Measuring interactions on protein arrays is performed in non-equilibrium conditions. This leads to loss of information and adversely affects the sensitivity of these methods. By using integrated microfluidics and applying a MITOMI, we were able to measure interactions at equilibrium. This led directly to higher sensitivity and for example for discovery of “weak” or transient interactions. Our efforts resulted in the discovery of new receptors to SV40 and HDV as well as new host targets for the HCV NS3 protease.
In MuDLOC, we have worked on several parallel aspects of the devices: We have invested a lot of energy on improving the fabrication process and the core platform design. We have bioengineered new assays that will serve as additional layers or dimensions to the platform. These include, DNA methylation, protein post translation modifications and tools for characterizing protein DNA binding.
For example, MuDLOC is now able to detect Tyrosine amino acid modification as well as Ubiquitination. These are very common modifications responsible for cellular communications and control of protein degradation, respectively. Moreover, MuDLOC can now distinguish between Tyr modifications that are self-inflicted (auto-phosphorylation) or made by a third party (regular phosphorylation).
The final goal in MuDLOC was a multi-dimensional platform that can study and characterize protein networks in vitro. All on a single platform, including protein-protein, protein-DNA and DNA or protein modifications. For example, we are now able to look at a DNA binding protein and characterize its genomic prevalence. We could also discover its protein regulators by interaction screening or its different modifications. We can characterize the effect of these regulations on the DNA binding properties of the protein. For example, study how different PTMs affect this regulator protein activity. This may lead to better and more accurate models predicting where and when the protein will bind on our genome. All these different experiments are performed on the same platform and under the same conditions.
The microfluidic fabrication facility we have built is now catering to many different scientist in Israeli and European academy. This way, we are using our fabrication knowledge and capabilities to disseminate the technology into the community.
We believe that MuDLOC is a fantastic platform for performing multi-dimensional proteomics. It has already made an impact in several areas from biochemistry to microbiology and to gene regulation. The productivity of MuDLOC is observed in the 16 peer-reviewed papers that were published during the project.
The advantages of this approach are many. For example, there is no need to purify proteins in advance. The method is also compatible with all protein types including, as we demonstrate, thousands of membrane proteins. Protein functionality is a serious drawback of most protein arrays, we demonstrated that the fresh proteins produced on the MuDLOC platform are functional. This is a significant improvement over previous protein arrays.
Measuring interactions on protein arrays is performed in non-equilibrium conditions. This leads to loss of information and adversely affects the sensitivity of these methods. By using integrated microfluidics and applying a MITOMI, we were able to measure interactions at equilibrium. This led directly to higher sensitivity and for example for discovery of “weak” or transient interactions. Our efforts resulted in the discovery of new receptors to SV40 and HDV as well as new host targets for the HCV NS3 protease.
In MuDLOC, we have worked on several parallel aspects of the devices: We have invested a lot of energy on improving the fabrication process and the core platform design. We have bioengineered new assays that will serve as additional layers or dimensions to the platform. These include, DNA methylation, protein post translation modifications and tools for characterizing protein DNA binding.
For example, MuDLOC is now able to detect Tyrosine amino acid modification as well as Ubiquitination. These are very common modifications responsible for cellular communications and control of protein degradation, respectively. Moreover, MuDLOC can now distinguish between Tyr modifications that are self-inflicted (auto-phosphorylation) or made by a third party (regular phosphorylation).
The final goal in MuDLOC was a multi-dimensional platform that can study and characterize protein networks in vitro. All on a single platform, including protein-protein, protein-DNA and DNA or protein modifications. For example, we are now able to look at a DNA binding protein and characterize its genomic prevalence. We could also discover its protein regulators by interaction screening or its different modifications. We can characterize the effect of these regulations on the DNA binding properties of the protein. For example, study how different PTMs affect this regulator protein activity. This may lead to better and more accurate models predicting where and when the protein will bind on our genome. All these different experiments are performed on the same platform and under the same conditions.
The microfluidic fabrication facility we have built is now catering to many different scientist in Israeli and European academy. This way, we are using our fabrication knowledge and capabilities to disseminate the technology into the community.
We believe that MuDLOC is a fantastic platform for performing multi-dimensional proteomics. It has already made an impact in several areas from biochemistry to microbiology and to gene regulation. The productivity of MuDLOC is observed in the 16 peer-reviewed papers that were published during the project.