Extensive secondary structures in nucleic acid targets seriously impede the binding of complementary oligonucleotide probes. We report here a method to conduct the detection under extremely low salt conditions where the secondary structures are less stable and more accessible. A new type of nanoparticle probes prepared by functionalizing gold nanoparticles with nonionic morpholino oligos is employed. Because of the salt-independent hybridization of the probes with nucleic acid targets, nanoparticle assemblies can be formed in 2 mM Tris buffer solutions containing 0-5 mM NaCl, leading to the colorimetric target recognition. The sharp melting transitions of the target-probe hybrids allow discrimination of single-base imperfection, including substitution, deletion, and insertion. The method works effectively in detecting sequences that are likely to form secondary structure. In addition, the study provides direct evidence of the relationship between the aggregate structure and the melting behavior of the DNA-linked nanoparticles.