| Grant Number: | 7R21GM072893-03 |
| PI Name: | MELLER, AMIT |
| PI Email: | ameller@bu.edu |
| PI Title: | ASSOCIATE PROFESSOR |
| Project Title: | Nanopore-FRET instrument for RNA folding analysis |
Abstract: DESCRIPTION (provided by applicant): A novel method to the kinetics of secondary structure formation of individual single-stranded DNA study (ssDNA) and RNA molecules is described. The method exploits the discovery that hairpin DNA molecules threaded through the approximately 1 nanometer pore made by the alpha-hemolysin membrane-channel can be unzipped in a controllable way by a voltage gradient. Information about the unzipping and re-zipping processes is obtained by the concurrent monitoring of the ion current flowing through the pore, and the Forster Resonance Energy Transfer (FRET) signals arising from labeled polynucleotides, during their transit through the pore. The unique combination of these two single-molecule methods in a robust instrument will enable the study of unfolding and refolding dynamics of DNA and RNA, ranging from the relatively simple case of a single hairpin structure to the study of the kinetics of ribozyme folding that contain multiple hairpins and pseudo knots. The folding kinetics of RNA, in general, and ribozymes in particular is difficult to measure because these molecules fold as they're transcribed. In the case of the HDV ribozyme an early bifurcation point is predicted to be responsible for the main misfolded path, which leads to the non-catalytic form. Biopolymer threading through the nanopore in conjunction with SM optical detection makes it possible to study sequential folding kinetics in general. Using this novel technique, we will study the kinetics of co-transcriptional folding of the HDV genomic ribozyme via the formation of the native and nonnative hairpins. The remarkably powerful capabilities of this novel method can directly be applied to a wide range of applications in molecular, cellular and genomic studies.
Thesaurus Terms:
chemical kinetics, fluorescence resonance energy transfer, nanotechnology, nucleic acid quantitation /detection, nucleic acid structure
electrical potential, genetic transcription, hemolysin, hepatitis D virus, ion transport, membrane channel, polynucleotide, ribozyme
confocal scanning microscopy, electrode
| Institution: | BOSTON UNIVERSITY |
| 881 COMMONWEALTH AVENUE | |
| BOSTON, MA 02215 | |
| Fiscal Year: | 2006 |
| Department: | BIOMEDICAL ENGINEERING |
| Project Start: | 01-FEB-2005 |
| Project End: | 31-JAN-2008 |
| ICD: | NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES |
| IRG: | ZRG1 |