Figure 1. Targeted nanoscale photobiotinylation using Femto-seq.

A. 4’-aminomethyltrioxsalen (4’-AMT) is a psoralen family DNA cross-linker, which forms covalent bonds primarily with thymidine residues when excited by UV or UV-like two-photon (2P) irradiation. The photoactivatable reagent used is 4′-Aminomethyltrioxsalen-PEG3-Biotin (AP3B), which enables covalent attachment of biotin to DNA. B. Absorbance spectra of AP3B. Use of 700 nm 2P excitation or longer UV (~360 nm) avoids shorter wavelength DNA absorptions. C. Two-photon absorption cross-sections of the AP3B photobiotinylation probe. D. The minimal volume (assuming a single XYZ spot) calculated for three different numerical aperture (NA) objectives. Using high NA, it is possible to biotinylate extremely small volumes within the nucleus. E. Streptavidin-Alexa 647 labeling of different sized photo-biotinylated regions (1x1, 2x2 and 4x4 pixel ROI box sizes) in a FISH-fixed U2OS nucleus after photo-biotinylation taken at 5 different cross-linking intensities. The 700 nm femtosecond pulses were delivered through 63x/1.4 Zeiss objective lens. Pixel size was 50 nm (5.3x zoom) and the pixel dwell time as 4.1μs and 30 passes over the ROI was used (123 μs per 0.0025 μm³) for all crosslinking powers shown. F. Lateral and axial dimensions a one-pixel region cross-linked using 6.28 mW at the above scanning parameters. G. Overview of the Femto-seq method (see text). H. U2OS transgene structure (adapted from ref. 3) used for Femto-seq validation experiments. I. U2OS cell after gene activation (+DOX) and IPTG removal to allow YFP-LacI binding for visualization of transgene site showing colocalization of the YFP-LacI and MCP-mCherry.