Periodic Reporting for period 1 - nanoEx (Towards nanopore proteomics: enhancing cytolysin performance through genetically encoded noncanonical amino acids)
Berichtszeitraum: 2020-04-01 bis 2022-03-31
Detection of low abundance or unstable proteins requires technology that is fast and highly sensitive. Nanopore biosensing is a rapidly evolving technology and one of the most attractive new techniques that enables protein identification at the single-molecule level. Facile sample preparation and portability of the MinION device make this technique ideal for in situ detection of proteins, increasing the chances of detecting unstable protein targets. This can be critical for biomarker or drug target discovery.
The main objective of this MSC action is to improve the properties of protein nanopores, which are important for their use in protein identification, through directed evolution and incorporation of noncanonical amino acids (ncAAs). For this purpose, two biological nanopores, an actinoporin and lysenin, were identified as suitable scaffolds. This research combines the researcher's knowledge of synthetic biology techniques, namely insertion of ncAA through genetic code expansion in E. coli, with the supervisor's expertise in natural cytolysins and nanopore biosensing. By integrating these individual areas of expertise, we were able to create stable cytolysin-derived nanopores with altered oligomerization parameters, modify the sensing region chemistry and diameter of these novel nanopores by incorporating five different ncAAs, obtain ncAA-containing nanopores with stable, low-noise open pore currents, and solve high-resolution structures of the engineered nanopores, thus achieving all of the scientific objectives outlined in the application.
In conclusion, the use of directed evolution approaches and the insertion of ncAAs can bring significant advances to nanopore biosensing. Nanopore-based detection of proteins is important for analysis of the human proteome and may be critical for identifying expression patterns and structure-function relationships that define healthy and pathological states.
The main objective of this MSC action is to improve the properties of protein nanopores, which are important for their use in protein identification, through directed evolution and incorporation of noncanonical amino acids (ncAAs). For this purpose, two biological nanopores, an actinoporin and lysenin, were identified as suitable scaffolds. This research combines the researcher's knowledge of synthetic biology techniques, namely insertion of ncAA through genetic code expansion in E. coli, with the supervisor's expertise in natural cytolysins and nanopore biosensing. By integrating these individual areas of expertise, we were able to create stable cytolysin-derived nanopores with altered oligomerization parameters, modify the sensing region chemistry and diameter of these novel nanopores by incorporating five different ncAAs, obtain ncAA-containing nanopores with stable, low-noise open pore currents, and solve high-resolution structures of the engineered nanopores, thus achieving all of the scientific objectives outlined in the application.
In conclusion, the use of directed evolution approaches and the insertion of ncAAs can bring significant advances to nanopore biosensing. Nanopore-based detection of proteins is important for analysis of the human proteome and may be critical for identifying expression patterns and structure-function relationships that define healthy and pathological states.
We engineered a novel actinoporin nanopore, which exhibits low noise, no gating, and can be readily inserted into polymer bilayers of MinION devices, making it an ideal candidate for in situ protein detection. Subsequently, this nanopore was evolved to form stable nonameric and even decameric pores in addition to canonical octamers. These alternative oligomerization states expand the range of possible protein analytes by simultaneously varying the pore diameter. We also discovered that these nanopores are particularly well suited for the detection of positively charged proteins, some of which serve as biomarkers for certain pathological conditions (patent pending).
We further introduced five different ncAAs into the evolved actinoporin and lysenin nanopores by genetic code expansion in E. coli, developed a protocol for the isolation of ncAA-containing nanopores, and characterized the activity of these proteins. Importantly, these ncAA-containing nanopores retain low noise in single-channel conductance measurements. The side-chain chemistries of the ncAAs used can be employed for biosensing purposes beyond protein sensing.
This work will be disseminated in at least three scientific publications (to be submitted by the end of 2022). The exploitation of the results includes a patent application, which will be filed in the coming months. The researcher received the ToxiconX Early Career Researcher award for this work, which was presented at the EUVEN 2021 conference. To date, three publications (one review, one original research paper and one whitepaper) have been published or accepted for publication. The researcher gave ten lectures to elementary and high school students during and following the Science is Wonderful! 2021 online event. She gave two lectures to students at University of Ljubljana and University of Zagreb on the principles of genetic code expansion and its applications. She supervised one student volunteer and one MSc student.
We further introduced five different ncAAs into the evolved actinoporin and lysenin nanopores by genetic code expansion in E. coli, developed a protocol for the isolation of ncAA-containing nanopores, and characterized the activity of these proteins. Importantly, these ncAA-containing nanopores retain low noise in single-channel conductance measurements. The side-chain chemistries of the ncAAs used can be employed for biosensing purposes beyond protein sensing.
This work will be disseminated in at least three scientific publications (to be submitted by the end of 2022). The exploitation of the results includes a patent application, which will be filed in the coming months. The researcher received the ToxiconX Early Career Researcher award for this work, which was presented at the EUVEN 2021 conference. To date, three publications (one review, one original research paper and one whitepaper) have been published or accepted for publication. The researcher gave ten lectures to elementary and high school students during and following the Science is Wonderful! 2021 online event. She gave two lectures to students at University of Ljubljana and University of Zagreb on the principles of genetic code expansion and its applications. She supervised one student volunteer and one MSc student.
This work combines synthetic biology techniques with the field of nanopore biosensing. By developing the novel actinoporin nanopore, we expand the collection of protein pores suitable for nanopore biosensing. We have shown that the genetic code expansion technique allows facile insertion of different side-chain chemistries into protein nanopores. Introducing ncAAs into nanopores could become a general strategy to adapt nanopores for the specific detection of different types of analytes.