COMPACTION MECHANISM OFINTERMEDIATE-SIZED DNAELUCIDATED BY FLUORESCENCELIFETIME CORRELATIONSPECTROSCOPY

摘要

In viruses, bacteria, prokaryotes, and sperm cells,DNA is packed into dense structures by positively chargedpolyamine molecules such as spermine. DNA packagingof single DNA molecules is called DNA compaction andcan be generated by a variety of multivalent cations. Thereis an impressive amount of studies characterizing the mor-phology of the compacted DNA state by cryoelectron mi-croscopy and it appears to be possible to rationalize theconditions for forming toroides, spherical globules, rods,or rackets. Single molecule fluorescence microscopy al-lowing to visualize large DNA molecules (166 kbp)clearly demonstrated the discrete ("all-or-non") characterof the DNA coil–globule transition. Moreover, Yoshi-kawa's group could visualize the compaction mechanismby fluorescence microscopy directly in real time3 andshowed that a compact part on a 166 kbp DNA chain ap-pears and grows within several seconds until completecollapse. The same group showed that under certain condi-tions these intermediate conformations can be stabilizedleading to unfolded and compact phases of DNA coexist-ing along a single DNA molecule. It is important to notethat all these mechanistic observation based on fluores-cence microscopy were done using rather large DNAmolecules. For example, for the bacteriophage T4 DNA 911 (166 kbp) that was preferentially used by the above men-tioned studies by Yoshikawa, a hydrodynamic radius ofabout 900 nm and 90 nm was determined for the unfoldedand spermine-induced compacted state, respectively4. Ina recent study5, fluorescence microscopy together witha Brownian motion analysis was for the first time appliedon the spermine-induced compaction of circular plasmideof an intermediate size (12.5 kbp). In contrast to thoselinear DNA molecules larger than several tens of kbp, theauthors found a comparable small change of the hydrody-namic radius from the elongated form (260 nm) to about150 nm in the compact state and characterized the foldingtransition as continuous. To our knowledge, this study isthe only single-molecule observation of compaction ofa non-adsorbed, freely diffusing circular plasmide of anintermediate size. Taking into account the physiologicalrelevance of such small" DNA structures5 and the factthat the resolution of the fluorescence microscopy used5 isless than 230 nm, there is certainly a need for applyingalternative techniques which are able to monitor on a sin-gle molecule level the spermine-DNA interaction in solu-tion. This motivated us to apply a recently developed sin-gle molecule fluorescence technique, fluorescence lifetimecorrelation spectroscopy (FLCS), for investigating thecompaction mechanism of a 10 kbp circular plasmide.