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Pulsed field gradient diffusion constant measurements were used to resolve the ambiguity in determining the conformational states of single-stranded DNA dodecanucleotides (d1s, d4s and d5s). For d1s and d5s, because of the spectral symmetry conventional NMR analyses cannot differentiate whether they are hairpins or homo-duplexes. However, the diffusion constants of these sequences at 300 K are 1.4 times greater than those of the comparison complementary duplexes. This result agrees well with what is expected for Dhairpin/Dduplex based on classic liquid-phase translational diffusion models and the Einstein-Stokes equation, confirming that d1s and d5s form hairpins. d4s did not show a structured spectral pattern, but its diffusion constant measurement suggests that this sequence may not be a random coil. The DNA sequences studied contain chemically modified backbone linkages and are potential antisense agents for gene regulation. The knowledge of their diffusion constants, in combination with conventional NMR analysis and other biophysical spectroscopic measurements, provides new insights into the relationships of chemical structure and conformational preference of antisense oligonucleotides and their analogs. |
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