Back

Splice-Aware Optimization Prevents Pervasive Missplicing of Natural and Synthetic cDNAs

Ahmad, M.; Bellido Molias, F.; Cano Aroca, L.; Mordstein, C.; Watson, S.; Bhuiyan, N.; Clarke, M. J.; Kimchi-Sarfaty, C.; Katneni, U.; Gaunt, E. R.; Hurst, L. D.; Netuschil, N.; Hofmeister, T.; Liss, M.; Kudla, G.

2026-05-25 synthetic biology
10.64898/2026.05.25.727620 bioRxiv
Show abstract

Heterologous gene expression is widely used across biology and medicine, and often relies on codon optimization to increase protein yields. Here we uncover missplicing as a common and largely unrecognized failure mode of heterologous expression. Using systematically designed libraries comprising over 5,000 synthetic reporter genes and natural human cDNAs, we find that the majority of gene variants expressed in a human cell line are at least partially spliced, and in many variants the spliced isoform dominates, reducing protein output or ablating expression entirely. By analysing sequence determinants of expression across multiple human cell lines, we uncover a hierarchical architecture of regulatory control, where GC content establishes baseline mRNA levels, local sequence features influence splicing, and tissue-specific codon adaptation to tRNA pools fine-tunes translation efficiency. These findings enable us to develop predictive models of expression and splicing, benchmark current optimization strategies, and design a splice-aware optimization algorithm that substantially improves transgene performance.

Matching journals

The top 3 journals account for 50% of the predicted probability mass.

1
Nature Communications
○ 5641 papers in training set
Top 5%
26.0%
Similar papers in this journal
2
Nucleic Acids Research
◐ 1281 papers in training set
Top 0.8%
18.1%
Similar papers in this journal
3
Nature Biotechnology
○ 172 papers in training set
Top 0.4%
7.1%
Similar papers in this journal
50% of probability mass above
4
Cell Systems
○ 201 papers in training set
Top 0.8%
5.4%
Similar papers in this journal
5
Nature Methods
○ 385 papers in training set
Top 2%
4.7%
Similar papers in this journal
6
Science
● 477 papers in training set
Top 2%
3.4%
7
Science Advances
● 1243 papers in training set
Top 11%
3.2%
8
Molecular Systems Biology
○ 162 papers in training set
Top 0.8%
2.7%
9
eLife
● 5828 papers in training set
Top 40%
2.6%
10
Proceedings of the National Academy of Sciences
● 2444 papers in training set
Top 21%
2.4%
11
RNA
● 189 papers in training set
Top 0.8%
2.1%
12
ACS Synthetic Biology
● 287 papers in training set
Top 1%
2.1%
13
Molecular Cell
○ 350 papers in training set
Top 3%
1.7%
14
Nature Genetics
○ 286 papers in training set
Top 3%
1.7%
15
Cell Reports
○ 1498 papers in training set
Top 24%
1.1%
16
Nature Structural & Molecular Biology
236 papers in training set
Top 3%
1.0%
17
Nature Chemical Biology
○ 119 papers in training set
Top 3%
1.0%
18
Cell
○ 431 papers in training set
Top 10%
0.8%
19
iScience
○ 1154 papers in training set
Top 42%
0.6%
20
Communications Biology
○ 993 papers in training set
Top 36%
0.6%
21
The EMBO Journal
○ 309 papers in training set
Top 8%
0.6%
22
Cell Reports Methods
○ 165 papers in training set
Top 5%
0.6%
23
Nature
○ 645 papers in training set
Top 12%
0.6%

"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.