Table 1.
Predicted fold and stability of hybridization interactions of RNA segments and experimental verification
| Segments in the two-step ligation | Predicted stability at 37°C |
Native PAGE | |
|---|---|---|---|
| Hybrid/Duplex (ΔG) | Intramolecular (ΔG) | ||
| G1-A8 + DNA splint | −4.8 (++, +) | NA | ND |
| G9-U28 + DNA splint | −22.5 (++, +) | NA | ND |
| G1-A8 | −1.8 | −0.5 | Duplexed |
| G9-U28 | −1.5 | −2.1 | >90% monomer, <10% duplexed |
| G1-U28 | −5.1 | −3.7 | Duplexed |
| G29-C61 | −6.3 | −9.6 | >90% monomer, <10% duplexed |
| G1-U28 + G29-C61 | −14.7 (+, +)a | NA | Hybridized |
| One-pot ligation | |||
| G1-A8 + G29-C61 | −4.3 (+, +)a | NA | No interaction |
| G9-U28 + G29-C61 | −7.4 (+, −)a | NA | No interaction |
| G9-C61 | Not formed | −14.9 (+, −) | ND |
| G9-C61 | Not formed | −12.9 (+, +) | ND |
| G1-A8 + G9-U28 + G29-C61 | Not predictable | NA | Smeared |
ΔG values are in kcal/mol at 37°C, 1 M NaCl, no Mg2+ and assuming a 10 μM strand concentration; (++, +), 100% base pairing, correct hybrid; (+, +), 50–100% base pairing, correct hybrid; (+, −), 50–100% base pairing, wrong hybrid; ND, not determined, NA, not applicable.
aPredicted structure displayed in Figure 3. Lowest values are given in bold for identification of the expected interaction.