Optical tweezers measurements were employed to directly observe viral DNA packaging in
wild type and packaging mutants of bacteriophage lambda. Several key findings are reported here:
DNA packaging by purified wild type lambda motors was measured for the first time, showing
nearly identical behavior in packaging DNA to crude extracts of terminase components. A slow
packaging lambda mutant, T194M, was found to package DNA at ~10× slower velocity than wild
type. Meanwhile another packaging mutant Y46F was found to package DNA slower than the wild
type (60-70% the velocity of the wild type velocity) as well as slipping >10x more frequently (per
length of DNA) than wild type. Another mutant (K84A) showed slower packaging (60-70% the
velocity of wildtype), but displayed slipping and pausing behavior similar to wild type. Finally the
pausing and slipping dependence on length of DNA packaged of the various terminases studied was
discovered, suggesting further structural defects of the mutants that are detrimental to translocation.
These studies confirm the location of an ATPase center in the N-terminal portion of gpA which is
responsible for translocation of dsDNA.
We have synthesized high quality type-II CdTe/CdSe near infrared quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer yielding quantum dots of narrow size distribution. Excitation and photoluminescence spectra reveal discrete type-II transitions, which correspond to energy lower that type-I bandgap. We have used a peptide coating technique on type-II and commercial near infrared quantum dots for delivery in live animals and cultured cells.
Colloidal NCs consist of an inorganic particle and an organic coating that determines
their solubility, functionality, and influences their photophysics. In order for these NCs to be
biocompatible, they must be water-soluble, nontoxic to the cell, and offer conjugation
chemistries for attaching recognition molecules to their surfaces. In addition they should
efficiently target to biomolecules of interest, be chemically stable, and preserve their high
photostability. The requirements for their application in single-molecule biological studies are
even more stringent: fluorescent NCs should be monodisperse, have relatively small size (to limit
steric hindrance), reduced blinking, large saturation intensity, and high quantum yield (QY).
We have developed a new functionalization approach for semiconductor nanocrystals based on a single-step exchange of surface ligands with custom-designed peptides. This peptide-coating technique yield small, monodisperse and very stable water-soluble NCs that remain bright and photostable. We have used this approach on several types of core and core-shell NCs in the visible and near-infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles. This peptide coating strategy has several advantages: it yields probes that are immediately biocompatible; it is amenable to improvements of the different properties (solubilization, functionalization, etc) via rational design, parallel synthesis, or molecular evolution; it permits the combination of several functions on individual NCs. These functionalized NCs have been used for diverse biomedical applications. Two are discussed here: single-particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals.