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In vitro selection of bacteriophage phi29 prohead RNA aptamers for prohead binding
1Department of Genetics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
The Journal of Biological Chemistry
|February 28, 1998
Summary
Bacillus subtilis bacteriophage phi29 prohead RNA (pRNA) is essential for DNA packaging. In vitro selection identified specific pRNA sequences that maintain DNA packaging activity, mimicking natural pRNA evolution.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Prohead RNA (pRNA) from Bacillus subtilis bacteriophage phi29 is crucial for in vitro DNA packaging.
- Specific regions of pRNA (residues 22-84) interact with the prohead portal vertex, the site of DNA packaging.
Purpose of the Study:
- To identify essential nucleotides in pRNA for prohead binding and DNA packaging.
- To discover biologically active pRNA variants through in vitro selection and evolution.
Main Methods:
- Randomization of pRNA segments to create diverse RNA libraries.
- In vitro selection (aptamer selection) for high-affinity prohead binding ligands.
- Competition binding assays, nucleotide sequencing, and DNA packaging assays to analyze pRNA mutants.
Main Results:
- Selected pRNA aptamers active in DNA packaging were predominantly wild-type after randomization of E stem/loop and C-E loop regions.
- Nucleotides G82 and G83 are critical for DNA packaging, forming intermolecular base pairs with C47 and C48 to create a pRNA hexamer.
- Mutant aptamers retaining full packaging activity had alterations in U residues at positions 81, 84, and 85 within the D loop.
Conclusions:
- In vitro selection processes closely mirrored the natural evolutionary pathway of pRNA.
- Specific nucleotide sequences and intermolecular interactions within pRNA are vital for efficient DNA packaging by bacteriophage phi29.