An improved 4'-aminomethyltrioxsalen-based nucleic acid crosslinker for biotinylation of double-stranded DNA or RNA

Abstract

Nucleic acid crosslinkers that covalently join complementary strands of DNA/RNA have applications in both pharmaceuticals and as biochemical probes. Psoralen is a popular crosslinking moiety that reacts with double stranded DNA and RNA upon exposure to longwave UV light. The commercially available compound EZ-link psoralen-PEG3-biotin has been used in numerous studies to crosslink DNA and double-stranded RNA for genome-wide investigations. Here we present a new probe, AP3B, which uses the psoralen derivative, 4'-aminomethyltrioxsalen, to crosslink and biotinylate nucleic acids. We show that AP3B is 4 to 5 times more effective at labeling DNA in cells and produces a comparable number of crosslinks with over 100 times less compound and less exposure to UV light in vitro than EZ-link psoralen-PEG3-biotin.

Fig. 1. Psoralen undergoes 2 + 2 cycloaddition reactions to form mono and bifunctional adducts with DNA.

Fig. 2. Structure of PP3B (A) and structure and synthesis of AP3B (B).

Fig. 3. Detecting DNA interstrand crosslinks with a denaturing agarose gel. Interstrand crosslinks result in shifting from low mass ssDNA to higher mass dsDNA bands. Both compounds show dependence on exposure to UV light. AP3B shows significant signal depletion of ssDNA bands at concentrations where PP3B has no apparent activity.

Fig. 4. Reaction rate comparison of PP3B and AP3B. (A) The concentrations of PP3B and AP3B in the treated samples were fixed at 2.5 mM and 20 μM, respectively. UV exposure time was between 1 and 30 min. (B) Quantification of reaction progress. The fluorescent intensities of the ssDNA and dsDNA bands were calculated to determine the percent of crosslinked DNA for each time point. 100% dsDNA indicates a complete reaction.

Fig. 5. Evaluation of the relative labeling efficiency of PP3B and AP3B in cells. (A) In cell fluorescence detection of biotinylation in Hela cells incubated with the indicated concentration of PP3B, AP3B or vehicle (10 μL DMSO). (B) Quantification of the nuclear rhodamine signal intensity with bars normalized the vehicle + UV intensity.