It has been known that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA [1, 15]. Therefore, we reasoned that DHX29 may enhance type I IFN signaling by recognizing the HMW Poly(I:C) and binding to MDA5. To test this possibility, we determined the ability of purified MDA5 and DHX29 to bind to HMW Poly(I:C). Indeed, we found that MDA5 interacted weakly to biotin-labeled HMW Poly(I:C), whereas DHX29 exhibited a strong HMW Poly(I:C)-binding ability, but not LMW Poly(I:C) (Fig 6D), suggesting that DHX29 is an RNA co-sensor. Importantly, co-incubation of MDA5 with DHX29 markedly enhanced the ability of MDA5 to bind to HMW Poly(I:C) (Fig 6D). To determine the DHX29 domain(s) required to enhance the HMW Poly(I:C)-binding capacity of MDA5, pull-down experiments were performed using purified full-length and truncated DHX29 proteins. Full-length DHX29, but not its truncated forms, enhanced the ability of MDA5 to bind to HMW Poly(I:C) (Fig 6E). Interestingly, full-length DHX29 and truncated DHX29c, but not DHX29a, DHX29b, or DHX29d, were pulled down with biotin-labeled HMW Poly(I:C) (Fig 6E), suggesting that DHX29c (containing the DEXD and helicase domains) has a strong HMW Poly(I:C)-binding ability. Taken together, these results suggest that DHX29 interacts with MDA5 through its N-terminus and binds to HMW Poly(I:C) through its DEXD and helicase domains, thus bringing MDA5 and HMW Poly(I:C) together and enhancing their interaction. These results may explain why full-length DHX29 is required to enhance the HMW Poly(I:C)-binding capacity of MDA5.