The disassembly, reassembly and stability of CCMV protein capsids.

Efficient procedures are described for the disassembly of Cowpea Chlorotic Mottle Virus (CCMV) into its viral-RNA and capsid-protein components, the separation of the RNA and protein, and the reassembly of the purified protein into higher order nanoscale structures. These straightforward biochemical techniques result in high yield quantities of protein suitable for further biophysical studies (AFM, X-ray scattering, NMR, osmotic stress experiments, protein phase-diagram) and nanotechnology applications (protein enclosed nanoparticles, protein-lipid nanoemulsion droplets). Also discussed are solution conditions that affect the stability of the self-assembled protein structure and explicitly show that divalent cation is not required to obtain stable protein structures, while the presence of even small amounts of Ba(2+) have a significant impact on protein self-assembly.

An unusual sugar conformation in the structure of an RNA/DNA decamer of the polypurine tract may affect recognition by RNase H.

Retroviral conversion of single-stranded RNA into double-stranded DNA requires priming for each strand. While host cellular t-RNA serves as primer for the first strand, the viral polypurine tract (PPT) is primer for the second. Therefore, polypurine tracts of retroviruses are essential for viral replication by reverse transcriptase (RT).

Enhanced stabilization of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21) and poly r(U:AU) by water structure-making solutes.

A variety of organic cations, cationic lipids, low molecular weight alcohols, sodium dodecylsulfate, trehalose, glycerol, low molecular weight polyethylene glycols, and DMSO were tested for their ability to modulate the stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21), poly r(U:A U) and their respective core duplexes, d(A-G)(6);d(C-T)(6), d(A)(21);d(T)(21), poly r(A-U). Very substantial enhancement of triplex stability over that in a physiological salt buffer at pH 7 is obtained with different combinations of triplex and high concentrations of these additives, e.g.

Stabilization of nucleic acid triplexes by high concentrations of sodium and ammonium salts follows the Hofmeister series.

The thermal stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6) and d(T)(21):d(A)(21);d(T)(21) was studied in the presence of high concentrations of the anions Cl(-), HPO(4)(2-), CH(3)COO(-), SO(4)(2-) and ClO(4)(-). Thermally-induced triplex and duplex transitions were identified by UV- and CD-spectroscopy and T(m) values were determined from melting profiles. A thermodynamic analysis of triplex transitions shows the limitations of commonly used treatments for determining the associated release or uptake of salt, solute or water.

Osmotic pressure inhibition of DNA ejection from phage.

Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres.