Risdiplam is the first approved small-molecule splicing modulator for the 
treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that 
risdiplam analogues have two separate binding sites in exon 7 of the SMN2 
pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding 
site. Importantly, the sequence of this GA-rich binding site significantly 
enhanced the potency of risdiplam analogues. In this report, we unambiguously 
determined that a known risdiplam analogue, SMN-C2, binds to single-stranded 
GA-rich RNA in a sequence-specific manner. The minimum required binding sequence 
for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using 
a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured 
spontaneous binding of a risdiplam analogue to the target nucleic acids. We 
uncovered, for the first time, a ligand-binding pocket formed by two sequential 
GAAG loop-like structures. The simulation findings were highly consistent with 
experimental data obtained from saturation transfer difference (STD) NMR and 
structure-affinity-relationship studies of the risdiplam analogues. Together, 
these studies illuminate us to understand the molecular basis of single-stranded 
purine-rich RNA recognition by small-molecule splicing modulators with an 
unprecedented binding mode.