Enzymatic attachment of proteins to fluorescent nanodiamond surfaces.

Fluorescent nanodiamonds are biocompatibleinfinitely photostableand fluoresce in a variety of wavelengthsincluding
the near infraredNIRregionThese characteristics suggest transformative applications in areas ranging from pure
biological research to biomedical imagingwhere protein conjugated nanodiamonds could be targeted to specific tissues
in living patients or even structures within individual cellsIn order to reach this potentialhoweverthe coupling of
specific targeting proteins to nanodiamond surfaces must be simplified to a point where rapid nanodiamond prototyping
can be carried out by personnel with little or no chemical conjugation expertiseTo address this needwe propose the
development of simplekitsfor the production of novel protein conjugated fluorescent nanodiamondswhere the end
user provides the recombinant targeting protein and specifies the nanodiamond core for conjugationThe kits would
consist of expression plasmids for production of a tagged version of the end user s target proteinalong with nanodiamondblanksfor quick and simple conjugationThe conjugation reactions will be mediated by either a naturally split transsplicing inteinor the Sortase A enzymeEach of these enzymatic coupling reactions allow uniform and highly oriented
binding of the target protein to the nanodiamond surface via either N terminalinteinor C terminalSortase Asinglepoint attachmentThusthe tagged target proteinprovided by the end user using their recombinant expression host of
choiceand nanodiamond blank will allow researchers with little skill in chemical protein conjugation to rapidly generate
highly customized nanodiamond prototypes for their desired applicationsIn our Phaseworkwe will develop two core
enzymatic coupling technologies based on the GOS TerL split inteinfor N terminal protein attachmentand the
Staphylococcus aureus Sortase A transpeptidasefor C terminal protein attachmentIn each casea short synthetic
peptide will be produced and chemically coupled to the nanodiamond surface using conventional methodswhile a
complimentary peptide will be genetically fused to the desired target protein using the provided expression plasmidsBoth the intein and Sortase reactions take place spontaneously under ambient conditionsallowing highly specific
coupling via simple mixing of the purified target protein and nanodiamond blankTo demonstrate these methodswe will
conjugate Green Fluorescent ProteinGFPandlactamase onto the surface of a suitable nanodiamondThese two wellcharacterized model proteins have been selected due to the availability of simple activity assayswhich will facilitate the
characterization and optimization of the coupling chemistriesIn the case of GFPwe will use confocal microscopy to
demonstrate co localization of the protein to the nanodiamond surfacewhilelactamase will allow demonstration of the
target protein retaining enzymatic activity after immobilizationIn additionthe coupling of both model proteins will be
quantified using a variety of conventional immunological and biochemical assay methodsThe generation of at least three
successfully coupled target protein configurations will be considered a successful demonstration of the PhasegoalsIn
future workwe will further develop the kit components and nanodiamond blank production processand demonstrate
additional applications of the system using FRET and more complex targeting and drug delivery strategies Project Narrative
We describe enzymatic processes for attaching proteins to fluorescent nanodiamonds for
bioimaging applicationsEnzyme mediated ligations are simplefastefficient and require little
chemical conjugation experience by the researcherThe resulting conjugates will have uniform
orientations and site specific linkages to the nanodiamond surfaceThese are much needed
improvements for the development of novel bioimaging agents and will enable the development
of a host of tools for the diagnosis and treatment of disease