Table 2. Splice donor site prediction.
The open reading frame of the SARS-CoV-2 Spike protein was analyzed by the Splice Site Predictor online tool SpliceRover (http://bioit2.irc.ugent.be/rover/splicerover). This online tool allows prediction of splice donor and splice acceptor sites. Predicted splice donor sites are provided in Table 1 for the SARS-CoV-2 wildtype Spike gene (A), for the codon-optimized Spike genes derived from the experimental Ad5.S vector, and the FDA/EMA-approved vector-based vaccines from AstraZeneca (Vaxzevria, ChAdOx1-S) and Janssen/J&J (Ad26.COV2.S), respectively. The amino acid coordinates of Spike protein domains S1, S2, and the minimal ACE2-binding domain are indicated.
| Position | Potential splice donor sites | Score | Type |
|---|---|---|---|
| A: Splice donor site prediction in wildtype Spike ORF | |||
| 454–473 | TGGATGGAAA•GTGAGTTCAG | 0.187 | +1 |
| 541–560 | GGAAAACAGG•GTAATTTCAA | 0.151 | +1 |
| 894–911 | GAAACAAAGT•GTACGTTGAA | 0.565 | +1 |
| 1323–1342 | TGATTCTAAG•GTTGGTGGTA | 0.357 | 0 |
| 1906–1925 | TATTCTACAG•GTTCTATTFT | 0.274 | +1 |
| 1996–2015 | ATTGGTGCAG•GTATATGCGC | 0.707 | +1 |
| 3296–3317 | GCACACACTG•GTTTGTAACA | 0.160 | +2 |
| Consensus | MAG•GTNNGTG | ||
| B: Splice donor site prediction in codon-optimized Spike ORF of Ad5 | |||
| 497–516 | GCACCTTCGA•GTACGTGTCC | 0.987 | +2 |
| 1175–1194 | TCACAAACGT•GTACGCCGAC | 0.187 | +2 |
| 1209–1228 | GGGAGATGAA•GTGCGGCAGA | 0.162 | 0 |
| 1812–1831 | CTCCAACCAG•GTGGCCGTGC | 0.540 | 0 |
| 2331–2350 | CACCCAAGAG•GTGTTCGCCC | 0.204 | 0 |
| 2949–2968 | ACTGGACAAG•GTGGAAGCCG | 0.318 | 0 |
| 2961–2980 | GGAAGCCGAG•GTGCAGATCG | 0.151 | 0 |
| 3083–3102 | AGATGTCTGA•GTGTGTGCTG | 0.318 | +2 |
| 3296–3315 | GCACCCATTG•GTTCGTGACC | 0.388 | +2 |
| 3452–3471 | AACTGGATAA•GTACTTTAAG | 0.443 | +2 |
| 3555–3574 | GCTGAACGAG•GTGGCCAAGA | 0.217 | 0 |
| 3605–3624 | AACTGGGGAA•GTACGAGCAG | 0.800 | +2 |
| C: Splice donor site prediction in codon-optimized Spike ORF of ChAdOx1-S | |||
| 497–516 | GCACCTTCGA•GTACGTGTCC | 0.986 | +2 |
| 1012–1030 | TTCGGCGAG• GTGTTCAATG | 0.266 | 0 |
| 1175–1194 | TCACAAACGT•GTACGCCGAC | 0.177 | +2 |
| 1209–1228 | GGGAGATGAA•GTGCGGCAGA | 0.188 | 0 |
| 1812–1831 | CTCCAACCAG•GTGGCCGTGC | 0.541 | 0 |
| 2331–2350 | CACCCAAGAG•GTGTTCGCCC | 0.215 | 0 |
| 2949–2968 | ACTGGACAAG•GTGGAAGCCG | 0.398 | 0 |
| 2961–2980 | GGAAGCCGAG•GTGCAGATCG | 0.273 | 0 |
| 3083–3102 | AGATGTCTGA•GTGTGTGCTG | 0.503 | +2 |
| 3296–3315 | GCACCCATTG•GTTCGTGACC | 0.381 | +2 |
| 3452–3471 | AACTGGATAA•GTACTTTAAG | 0.245 | +2 |
| 3555–3574 | GCTGAACGAG•GTGGCCAAGA | 0.310 | 0 |
| 3605–3624 | AACTGGGGAA•GTACGAGCAG | 0.686 | +2 |
| D: Splice donor site prediction in codon-optimized Spike ORF of Ad26.COV2.S | |||
| 563–582 | ACCTGCGCGA•GTTCGTGTTC | 0.150 | +2 |
| 1175–1194 | TCACAAACGT•GTACGCCGAC | 0.180 | +2 |
| 1209–1228 | GGGAGATGAA•GTGCGGCAGA | 0.207 | 0 |
| 1812–1831 | CAGCAATCAG•GTGGCAGTGC | 0.547 | 0 |
| 2331–2350 | CACCCAAGAG•GTGTTCGCCC | 0.202 | 0 |
| 2961–2980 | TGAGGCCGAG•GTGCAGATCG | 0.194 | 0 |
| 3083–3102 | AGATGTCTGA•GTGTGTGCTG | 0.495 | +2 |
| 3296–3315 | GCACCCATTG•GTTCGTGACA | 0.392 | +2 |
| 3452–3471 | AACTGGACAA•GTACTTTAAG | 0.436 | +2 |
| S1 ectodomain: encoded by nucleotides 1–2049 (aa 1–683) | |||
| S2 domain: encoded by nucleotides 2050–3822 (aa 684–1273) | |||
| ACE2-binding domain: encoded by nucleotides 998–1720 (aa 319–551) | |||