Direct activation of SARM1 by dsDNA is not supported by biochemical and cellular evidence.

Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is a NAD+-consuming enzyme that drives axon degeneration and is activated by changes in the NMN/NAD+ ratio. A recent study proposed that cytosolic double-stranded DNA (dsDNA) directly binds SARM1's TIR domain, activating it independently of this canonical mechanism. Here, we evaluate dsDNA-dependent SARM1 activation using purified SARM1, biochemical and biophysical assays, primary neurons, and cellular pharmacology. Across multiple platforms, dsDNA does not stimulate SARM1 NADase activity, promote higher-order assembly, or generate the metabolic signature of activated SARM1. When effects are observed, they are weak, context-dependent, and inconsistent across assays, and do not support a defined activation mechanism. In cells, dsDNA induces NAD+ depletion through PARP-dependent pathways independently of SARM1. The reported DNA-binding mutant (3KE-SARM1) is nonfunctional in canonical SARM1 activation paradigms such as axotomy and therefore cannot be used to infer dsDNA activation mechanisms. Together, these findings do not support a model in which SARM1 is directly activated by dsDNA.