PROBING ROTATION AND REACTION OF BIOMOLECULES CONFINED TO SUB-FEMTOLITER VOLUMES

Jianyong Tang, Rani B. Kishore, Ana Jofre, Mark, E. Greene, Nathan Hodas, Kristian Helmerson, Lori S. Goldner

Abstract:

We measure the rotational and chemical dynamics of molecules confined to sub-femtoliter aqueous droplets that can be optically trapped (“hydrosomes’).  In small volumes, as in the confining environment of cells and organelles, it is necessary to ask what the effect of the boundaries and confinement are on the physical and chemical dynamics of the contained molecules. We begin to answer this question by measuring the rotational dynamics of confined molecules, including proteins, DNA and RNA.  Time resolved fluorescence anisotropy is used as a diagnostic for tumbling rates of molecules.  For most water-soluble molecules investigated, we find the tumbling rates inside the hydrosome are similar to those found in bulk solution.  However, we do find that surfactants play a crucial role in maintaining the integrity of confined biomolecules, in particular for proteins such as EGFP. To study biochemical reactions in the confining environment of hydrosome, we have developed and report on an automated dual micro-injector based platform for generating, mixing, and interrogating the contents of hydrosomes.  Two sub-femtoliter aqueous droplets, which contain biomolecules of different reagents, can be generated on demand from a dual-injector apparatus and then fused, which facilitates assembly of two separate moities to form a molecular complex.  In a typical experiment, hydrosomes are injected and then, by computerized control of a piezoelectric translation stage and single particle video tracking techniques, automatically trapped and mixed by dual optical tweezers to trigger a reaction process with precise timing. We demonstrate application of this droplet-mixing technique to study the reactions of calcium/fluo-3 binding and protein/DNA interactions.

Author:  Jianyong Tang

Mentors: Lori S. Goldner and Kristian Helmerson

Division: Atomic Physics Division

Laboratory: Physics Laboratory

Office: Instrument East (216), Room B217

Mail Stop: 8424

Email: jtang@nist.gov

Tel: 301-975-4485

Fax: 301-975-6991

Not a Sigma Xi member

Category: Biotechnology