DNA aptamers have been recently applied as simple and fast colorimetric sensors for a wide range of molecules. A unique feature of these systems is the presence of non-base pairing oligonucleotides in both DNA aptamers and spacers on DNA-functionalized nanoparticles. We report here a systematic investigation on an adenosine aptamer-linked gold nanoparticle system. When the aptamer overhang and the spacer were aligned on the same side, adenosine-responsive disassembly was inhibited. This inhibition effect increased with the length of the spacer, and fully inhibited activity was observed with the spacer containing more than three nucleotides. In contrast to a linear relationship between the spacer length and melting temperature in double-stranded DNA systems without overhangs, the aptamer system displayed a nonlinear relationship, with the melting temperature decreasing exponentially with spacer length. Control experiments suggested that this inhibition effect was due to thermodynamic factors rather than kinetic traps. A comparison with aptamer beacon systems indicated that nanoparticles may play an important role in this inhibition effect, and no specific interactions between the aptamer overhang and spacer were detected. The identity of nucleotides in the spacer did not affect the conclusions. Furthermore, the rate of disassembly or color change was slower at lower temperature or higher ionic strength, but was little affected by pH from 5.2 to 9.2. Therefore, non-base pairing DNA provided another dimension for controlling DNA-linked nanoparticles in addition to pH, temperature, or ionic strength, and this knowledge has resulted in the most optimal construct for sensing applications.