This study investigates the origin and structural evolution of de novo genes in Drosophila. Using high-quality whole-genome alignments and computational structural modeling, researchers identified 555 de novo gene candidates in D. melanogaster that originated within the Drosophila lineage. These genes show gradual shifts in sequence composition, evolutionary rates, and expression patterns with their gene ages, suggesting possible functional or adaptive changes. However, there are few overall protein structural changes in these candidates. Some candidates have potentially well-folded protein structures, and ancestral sequence reconstruction analysis shows that most of these are well-folded in their ancestral stages. Single-cell RNA-seq analysis in testis shows that while most de novo gene candidates are enriched in spermatocytes, several are biased towards early spermatogenesis, indicating the potential importance of early germline cells in de novo gene origination. De novo genes are novel genes born from non-genic DNA sequences. Recent studies suggest that de novo genes may not necessarily be disordered and could have conserved structures after origination. Despite advancements, understanding the protein structures of de novo genes remains limited. AlphaFold2 and ESMFold were used to evaluate foldability, showing that most de novo genes are partially folded or not folded, with only a few potentially well-folded. Structural stability and dynamics were analyzed using molecular dynamics simulations, revealing that de novo genes may undergo gradual sequence and functional changes without significant structural changes. The origin of Drosophilae-specific de novo genes is more likely associated with open chromatin than transposable elements. De novo gene candidates show biased expression in the testis, head, and ovary, and are associated with open chromatin regions. De novo genes are mostly adaptive and shaped by both adaptive and non-adaptive changes. They tend to be more disordered, more exposed, and more likely to be transmembrane or secretory proteins. De novo genes have higher male specificity and tissue specificity, and lower expression levels in females. De novo gene candidates undergo gradual sequence/function changes without significant structural changes. Most de novo genes are partially folded or not folded, with only a few potentially well-folded. These well-folded genes may have complex structural folds. Ancestral sequence reconstruction analysis shows that most well-folded de novo genes are born well-folded. Early germline cells in the testis play a non-negligible role in de novo gene origination, despite most de novo genes being enriched in later germ cells. The study provides a systematic overview of the origin, evolution, and protein structural changes of Drosophila-specific de novo genes. It highlights the potential for de novo proteins to be well-folded and even folded into novel protein folds. The findings suggest that de novo genes may have important molecular functions with certain fitness effects, partly due to their tendency to encode signal proteins or transmembrane proteins. The study also indicates that de novo genes may retain certain basic molecular functions byThis study investigates the origin and structural evolution of de novo genes in Drosophila. Using high-quality whole-genome alignments and computational structural modeling, researchers identified 555 de novo gene candidates in D. melanogaster that originated within the Drosophila lineage. These genes show gradual shifts in sequence composition, evolutionary rates, and expression patterns with their gene ages, suggesting possible functional or adaptive changes. However, there are few overall protein structural changes in these candidates. Some candidates have potentially well-folded protein structures, and ancestral sequence reconstruction analysis shows that most of these are well-folded in their ancestral stages. Single-cell RNA-seq analysis in testis shows that while most de novo gene candidates are enriched in spermatocytes, several are biased towards early spermatogenesis, indicating the potential importance of early germline cells in de novo gene origination. De novo genes are novel genes born from non-genic DNA sequences. Recent studies suggest that de novo genes may not necessarily be disordered and could have conserved structures after origination. Despite advancements, understanding the protein structures of de novo genes remains limited. AlphaFold2 and ESMFold were used to evaluate foldability, showing that most de novo genes are partially folded or not folded, with only a few potentially well-folded. Structural stability and dynamics were analyzed using molecular dynamics simulations, revealing that de novo genes may undergo gradual sequence and functional changes without significant structural changes. The origin of Drosophilae-specific de novo genes is more likely associated with open chromatin than transposable elements. De novo gene candidates show biased expression in the testis, head, and ovary, and are associated with open chromatin regions. De novo genes are mostly adaptive and shaped by both adaptive and non-adaptive changes. They tend to be more disordered, more exposed, and more likely to be transmembrane or secretory proteins. De novo genes have higher male specificity and tissue specificity, and lower expression levels in females. De novo gene candidates undergo gradual sequence/function changes without significant structural changes. Most de novo genes are partially folded or not folded, with only a few potentially well-folded. These well-folded genes may have complex structural folds. Ancestral sequence reconstruction analysis shows that most well-folded de novo genes are born well-folded. Early germline cells in the testis play a non-negligible role in de novo gene origination, despite most de novo genes being enriched in later germ cells. The study provides a systematic overview of the origin, evolution, and protein structural changes of Drosophila-specific de novo genes. It highlights the potential for de novo proteins to be well-folded and even folded into novel protein folds. The findings suggest that de novo genes may have important molecular functions with certain fitness effects, partly due to their tendency to encode signal proteins or transmembrane proteins. The study also indicates that de novo genes may retain certain basic molecular functions by