|M.Sc Student||Marbach Daphna|
|Subject||A Nanopore Tool for Single-Molecule Analysis of|
|Department||Department of Biomedical Engineering||Supervisor||PROF. Amit Meller|
|Full Thesis text|
MicroRNAs (miRNAs) are short noncoding single stranded RNAs that are key posttranscriptional regulators of gene expression. miRNAs regulate protein synthesis by base-pairing to the miRNA-recognition-elements of the target mRNAs, which are usually located in the mRNA 3’UTR. miRNAs dysfunction may result in a broad spectrum of diseases. Despite their importance, the mechanistic details of miRNA function remain to date poorly understood.
In the present study, nanopore technique is used for the first time in order to study the mechanistic details of miRNA regulation, at the single-molecule level. In particular, the experiments conducted as part of this study, are the first step towards the analysis of mRNA-miRNA interactions, using the α-Hemolysin (a-HL) nanopore.
In a nanopore experiment, a strong electrical field is used to force charged biopolymers (e.g. - ssDNA and ssRNA) through a single pore in a single file manner. Sequential single-molecule passages through the pore can be characterized and yield information that is normally masked by the ensemble averaging in bulk techniques, such as bond strength. The a-HL nanopore is used in the present study due to its inner diameter of only 15 Å that allows resolving single-stranded nucleic acids.
The biological model chosen for the present study is human PTEN’s mRNA and human miR-29a. Previous studies have experimentally verified that miR-29a targets PTEN through two known target sites located on the PTEN 3’UTR. Of these two target sites this study will focus only on the first site.
Several different segments of PTEN’s 3’UTR with lengths ranging from ~50 to ~800 bases, incorporating the miR-29a 1st target site in their middle or their end, were synthesized. These segments were synthesized using PCR followed by In Vitro Transcription. Since these segments are later used in single-molecule nanopore experiments their high purity is essential. Therefore they were purified by employing a protocol that was especially developed as part of this study.
An experimental setup was assembled and calibrated using two chemically synthesized ssDNAs: poly(AC)40 and A30C30A30. Translocation experiments were conducted on these two biopolymers and the obtained results were found to be in line with previously published results.
Translocation experiments were conducted on two of the PTEN 3’UTR ssRNA segments that were biologically synthesized as part of this study (lengths of ~200 and ~800 bases). These segments translocations showed a richer pattern than that obtained for the translocations of the chemically synthesized segments. A preliminary model for this pattern was suggested.