DART User Guide

Guidelines for DART

The DART technology is a novel approach for targeting neuropharmaceuticals to endogenous receptors of different cell types in the brain.  The tool consists of a 2-part system:

  1. an AAV vector to generate HaloTag protein (HTP) expression on the surface of your cell type of interest
  2. a DART chemical to covalently tether the drug or dye payload to the cells expressing the HTP and allow for endogenous receptor interaction

AAV strategy

DART relies on strong expression of the HTP without toxicity in the cell type of interest; thus optimization of the viral strategy will be a key factor to successful DART experiments. The AAVs are Cre-dependent to allow for robust, directed expression in a variety of cell types found in traditional Cre mouse lines.

DART math

Each DART is provided as a highly-soluble pure compound. From these pure compound aliquots, different concentrations are created depending on the volume of diluent added to the vial.  Your specific assay needs will determine which size aliquot you use.  (see Shields et al., 2024 NatureMethods for more information)

In an in-vitro study, incubation in a 300nM (nanoMolar) solution of DART for at least 15min at room temperature is enough to saturate all of the active HTPs in a sample.  Therefore, for example:

  • For ex-vivo slice electrophysiological: a 30nm vial in a total of 100mL of base solution (final concentration = 300nM), with a flow of 15mL of DART solution over a 15min time course (i.e., 1mL/min rate) would allow enough DART solution for approximately 5 slices from one 30nm aliquot.
  • For in-vitro cultured samples or for pre-incubation (used for competitive antagonist DARTs): a 3nm vial would provide a total of 10mL DART solution at 300nM final concentration, which can be used for multiple coverslips or slices over the course of 1 day
  • For in-vivo uses: given the small 1uL volume restraint for direct brain infusions, typically a 3nm vial is mixed with 100uL of aCSF (or other suitable physiological solution) to create a higher concentration (30uM final) to allow slow diffusion capture over a period of time.  For lateral ventricle infusions, a larger 300uM concentration bolus is recommended (1uL/hemisphere at 300uM drug-DART or 300uM PEG+300uMblank+30uM dye).

Helpful considerations

 - Use a hyperosmotic PBS diluent for virus dilutions

We highly recommend using a hyperosmotic PBS for virus dilution, especially for the VB (VectorBuilder) virus. A high-salt PBS supplemented with additional 200mM NaCl (ie, 0.59g NaCl in 50mL PBS then filtered thru a 0.22um CA cellulose acetate membrane and stored at 4c) seems to help with potential AAV particle aggregation and reduce expression variability.

 - In-vivo volumes & concentrations may differ depending on your area of interest

Different regions of the brain might need different dilutions based on density/volume of your target tissue. For example, the large striatum usually needs ~ 30uM concentration for direct 1uL infusions, but the smaller VTA area might only need ~3-10uM.

 - Vectashield Vibrance for histology

We strongly recommend using Vectashield Vibrance media (with or without DAPI; Vector Labs) for histological mounting and coverslipping of brain sections. This specific mounting media maintains the highest level of fluorescence of the Alx647.1-DART.2 dye during the imaging process, allowing clear identification of the location of DART capture for post-hoc confirmation and analysis.

https://www.biorxiv.org/content/10.1101/393264v2 for more information

 - Discard excess reconstituted DART at the end of the day

Though we have promising pilot data suggesting that the DART molecules are stable at 37c for prolonged periods of time, we have not yet fully characterized the extent of DART stability. Therefore, we encourage making a fresh aliquot of DART for each day of experiments, maintain the aliquot on ice until use, and then discard any extra reconstituted DART at the end of the day.