Optimization of antisense drug design against conservative local motif in simulant secondary structures of HER-2 mRNA and QSAR analysis
Abstract
AIM: To study the role of mRNA secondary structure stability in antisense drug design and obtain better antisense candidates against neu/HER-2/erbB-2 mRNA than previous report.
METHODS: Program RNAstructure was utilized to simulate the secondary structures of HER-2 mRNA. Then 21 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting different parts of secondary structural motif were designed. HA4 was set as positive control. Mean 50 % inhibitory effects (IC(50)) of S-ODN on proliferations of SK-BR-3 breast cancer cells were evaluated. The expression of target mRNA was detected by RT-PCR. The multiple regression and quantitative structure-activity relationship (QSAR) analysis was preformed by SPSS software.
RESULTS: One optimal and two suboptimal secondary structures of target mRNA were obtained. Nine out of 11 S-ODN against completely conservative local motif (LM) (conservative among all simulant secondary structures) got lower or similar IC(50) values compared with HA4. On the other hand, 2 out of 3 S-ODN against relatively conservative LM (conservative between any two simulant secondary structures) got lower or similar IC(50) values compared with HA4. Only 2 out of 5 S-ODN targeting variable LM (variable among different predicted secondary structures) had acceptable activities. Average IC(50) of S-ODN against completely conservative LM was significantly lower than that of S-ODN against diverse LM. QSAR analysis suggested that stability, base number of bulge loops, and target free energies Delta GoT were statistically significant. In the multiple regression, R was 0.967, P=0.005.
CONCLUSION: Antisense drug design against conservative LM was helpful for improving the positive rate. Several S-ODN candidates better than positive control were screened.
Keywords:
METHODS: Program RNAstructure was utilized to simulate the secondary structures of HER-2 mRNA. Then 21 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting different parts of secondary structural motif were designed. HA4 was set as positive control. Mean 50 % inhibitory effects (IC(50)) of S-ODN on proliferations of SK-BR-3 breast cancer cells were evaluated. The expression of target mRNA was detected by RT-PCR. The multiple regression and quantitative structure-activity relationship (QSAR) analysis was preformed by SPSS software.
RESULTS: One optimal and two suboptimal secondary structures of target mRNA were obtained. Nine out of 11 S-ODN against completely conservative local motif (LM) (conservative among all simulant secondary structures) got lower or similar IC(50) values compared with HA4. On the other hand, 2 out of 3 S-ODN against relatively conservative LM (conservative between any two simulant secondary structures) got lower or similar IC(50) values compared with HA4. Only 2 out of 5 S-ODN targeting variable LM (variable among different predicted secondary structures) had acceptable activities. Average IC(50) of S-ODN against completely conservative LM was significantly lower than that of S-ODN against diverse LM. QSAR analysis suggested that stability, base number of bulge loops, and target free energies Delta GoT were statistically significant. In the multiple regression, R was 0.967, P=0.005.
CONCLUSION: Antisense drug design against conservative LM was helpful for improving the positive rate. Several S-ODN candidates better than positive control were screened.