Dr. Ana Jofre – National Institute of Standards and Technology
“HYDROSOMES: Optically trapped femtoliter containers for single molecule studies”
I will discuss a novel technique for creating, manipulating, and combining femtoliter volume chemical containers. Possible uses include creating controlled chemical reactions involving small quantities of reagent, and studying the dynamics of single transiently interacting molecular complexes. The containers, which we call hydrosomes, are stable aqueous droplets in a low index-of-refraction fluorocarbon medium. The index of refraction mismatch between the container and fluorocarbon is such that individual hydrosomes can be optically trapped by single focus laser beams, i.e. optical tweezers. Studies of single molecule dynamics on time scales > 1 ms often involve surface-attachment of the molecule to prevent it from leaving the detection region. Here we obviate the need for both the surface and surface attachment chemistries by confining the molecule to a hydrosome that is optically immobilized in the detection volume of a confocal microscope. Polarization anisotropy studies suggest that hydrosome encapsulated molecules have full rotational freedom within the hydrosome. Furthermore, measurements of fluorescence resonance energy transfer (FRET) from donor and acceptor dyes attached to single RNA 16mers suggests that there is a significant perturbation from the PEG tether used to immobilize the RNA on the surface that is absent for the hydrosome encapsulated RNA. Hydrosome encapsulation presents an important advantage of over liposome encapsulation techniques in that hydrosomes fuse on contact, thereby facilitating mixing of the encapsulated components. The various molecular components of a complex can be isolated and confined in different hydrosomes, which can then be fused to form a single larger hydrosome containing all elements of the complex. Our intent is to use hydrosome encapsulation and mixing to perform studies of transiently interacting complexes, such as RNA/protein systems. Systems that will be described and discussed include the R1inv-R2inv/Rom interaction relevant to the formation and inhibition of the ColE1 primer.