Active droplets, membraneless compartments driven by internal chemical reactions, are compelling models for protocells and synthetic life. A central challenge is to program their dynamic behaviors using heritable genetic information, which would grant them the capacity to evolve. Here, we create transiently active RNA droplets by integrating sites for ribozyme catalysis directly into the sequence of self-assembling, four-arm RNA nanostars. To enable perfusion and observe the resulting dynamics over time, we develop a method for trapping individual droplets in hydrogel cages by targeted in situ photopolymerization. This enables us to quantify the sequence-programmable droplet dissolution and to control the degradation kinetics by choosing between fast (hammerhead) and slow (hairpin) ribozymes. Furthermore, we trigger the segregation of mixed droplet populations via the sequence-specific cleavage of a chimeric linker RNA. The droplet-encapsulated DNA templates code for the regrowth of new droplets, establishing the proof-of-principle for a minimal, genetically encoded cycle of dissolution and regrowth. By directly linking RNA sequence to droplet stability, composition, and life-cycle dynamics, our work provides a robust platform for engineering evolvable materials and advancing the bottom-up construction of syntheticcells.

Zygotic genome activation (ZGA) failure leads to developmental arrest and poses a clinical challenge to women's fertility. We observed that human embryos arresting at the eight-cell ZGA stage exhibited specific down-regulation of endogenous retrovirus MLT2A1. Depleting MLT2A1 resulted in a failure in embryo development and a reduction in ZGA gene expression. Mechanistically, MLT2A1s synthesized chimeric transcripts with downstream coding and noncoding sequences, predominantly with heterologous retro-transposable elements. These diverse fusion sequences expanded the genome-targeting spectrum of MLT2A1 RNAs. Nevertheless, the shared MLT2A1 sequences partnered with heterogeneous nuclear ribonucleoprotein U (HNRNPU) to recruit RNA polymerase II, promoting global transcription of ZGA genes and autoamplification of the MLT2A1 subfamily. Thus, MLT2A1 chimeric RNAs formed an interlocking network that acts synergistically to boost human ZGA and early embryogenesis.

Abstract Chromosomal translocations are commonly harbored in rare mesenchymal cancers known as soft tissue sarcomas (STS). These translocations often result in functional oncogenic fusion proteins which are pathognomonic for some STS. To study the transcriptional consequences of these aberrant chimeras across STS, we performed deep RNA sequencing on 39 patent samples with suspected fusion oncoproteins. Samples are representative of Ewing sarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, myxoid/round cell liposarcoma, endometrial stromal sarcoma, and synovial sarcoma. Unexpectedly, we identified many unique and shared chimeric RNA across all samples including pathognomonic EWSR1 containing fusions as expected for specific subtypes. In Ewing sarcoma (EwS), EWSR1-FLI1 is the most common diagnostic fusion, which can occur via a balanced or complex translocation. Where a balanced translocation occurs, expression of both the canonical fusion (EWSR1-FLI1) and the reciprocal fusion (FLI1-EWSR1) may be seen. However, relatively little is known about the reciprocal fusion. Upon induction of a reciprocal construct in the EwS cell line A673, we identified differential gene expression of the noncoding RNAs including RN7SK, RN7SL1, RN7SL2, and RPPH1. RN7SK is a small nuclear RNA that regulates gene transcription by interacting with the positive transcription elongation factor b (P-TEFb) and plays a role in stem cell proliferation, differentiation, senescence, and apoptosis. Interestingly, noncoding RNA fusions with RN7SK chimera involving RNA pol III transcripts were the most abundant fusions identified across all STS tumors, though a functional role for these aberrant transcripts has not been defined. Wild type EWSR1 plays a role in RNA splicing, and these data suggest that EwS and EwS fusions may play a role in the regulation of ncRNA fusions and subsequently the disruption of normal RN7SK regulatory function. We aim to evaluate whether ncRNA fusions can also be identified in non-fusion driven sarcomas, at what point such ncRNA fusions may be arising in the central dogma, and what their function may be in sarcomagenesis. Further study regarding the function and consequences of these RNA fusions is required to understand their importance in disease initiation and progression. Citation Format: Emily Isenhart, Sarah Gawlak, Kristen Humphrey, Michalis Mastri, Scott Olejniczak, Miranda L. Lynch, Joyce Ohm. ConFusion: The unexpected presence of non-coding RNA fusions in fusion-positive soft tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B023.

Abstract Introduction: In Non-Small Cell Lung Cancer (NSCLC), low-fidelity transcription mechanisms such as ecDNA (extra-chromosomal DNA) are an increasingly relevant aspect of progression and prognosis, warranting comprehensive investigation into stability and prevalence of chimeric RNA transcripts. Here, we describe global distribution of fusion transcripts in encounter-matched primary tumor and lymph node metastases and their prognostic implications. Methods: RNAseq was performed on resected FFPE (encounter-matched primary and lymph nodes) from 121 stage IIB-IIIA (T1-4N1-2M0) NSCLC patients, in addition to 693 Stage IA1-IB cases (T1a-2aN0M0). STAR and STAR-Fusion were used to align data to the GRCh37 genome. Differential gene expression was based on specimen source (primary or lymph node). Aggregate analyses were also performed, including determination of a fusion burden metric, assessing the relationship between primary tumors, lymph node metastases, and recurrence. Deconvolution of Tumor Microenvironment was performed via Quantiseq. Results: Comparing primary tumors to paired lymph node metastases, 7,930 ensembl IDs were differentially-expressed at FDR < 0.001 (n=101 pairs). A total of 613 fusions (685 unique transcripts) were detected from paired primaries and lymph node metastases (n = 242). Frequency of specific gene fusions in primaries ranged from 0-0.963 (median 0.019), compared to 0-0.870 (median 0.0093) in LN metastases (p=1.87×10-4). We observed fusion of TULP4-RP11-732M18.3 as pervasive in NSCLC, occurring in 84-87% of primary tumors and lymph nodes, with a 91% retention rate. Conversely, fusions involving CCDC7 or SEPT14 were preferentially observed in metastases, where 82% of CCDC7 fusions and 75% of SEPT14 fusions were not observed in the primary tumor despite occurring at high frequency (66% and 18.5%, respectively) among LN metastases. Interestingly, median fusion burden (unique gene-gene fusions) for primaries was 14 compared to 11 (p=2.06×10-7) in LN metastases (median change -4). Among N0 cases that did not receive adjuvant therapy, median fusion burden in non-recurrent tumors was 15 (n=570), compared to 13 in those that recurred over 3-60 months (n=123; p = 0.04). Interestingly, there was no significant difference in fusion burden between recurrent cases and primary tumors with LN metastases (n = 108). For both recurrent cases and LN-paired primaries, the difference in fusion burden compared to LN metastases (n = 110) was significant (p=2.10×10-4 and 2.06×10-7). A subset of LN metastases (n=27) increased in fusion burden relative to the primary tumor; these primaries uniquely exhibited 33% decrease in regulatory T cell content (p=0.008), 20% decrease in M2 macrophage content (p = 0.01 with M1 macrophages static), and 40% decrease in B-cell content (p=0.05). Conclusions: Fusion transcripts are pervasive in RNAseq data, suggesting a broader and more variable landscape than accounted for by primary tumors alone. Concurrent ongoing studies explore the role of ecDNA in fusion burden changes related to NSCLC progression and metastasis. Citation Format: Kelly M. Cagin, Keri L. Denson, Sierra R. Broad, Wara M. Naeem, Arsalan M. Khan, Michael M. Liptay, Christopher W. Seder, Jeffrey A. Borgia. A global analysis of fusion transcripts and recurrence in NSCLC primary-lymph node pairs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B027.

Abstract Active droplets, membraneless compartments driven by internal chemical reactions, are compelling models for protocells and synthetic life. A central challenge is to program their dynamic behaviors using heritable genetic information, which would grant them the capacity to evolve. Here, we create transiently active RNA droplets by integrating sites for ribozyme catalysis directly into the sequence of self‐assembling, four‐arm RNA nanostars. To enable perfusion and observe the resulting dynamics over time, we develop a method for trapping individual droplets in hydrogel cages by targeted in situ photopolymerization. This enables us to quantify the sequence‐programmable droplet dissolution and to control the degradation kinetics by choosing between fast (hammerhead) and slow (hairpin) ribozymes. Furthermore, we trigger the segregation of mixed droplet populations via the sequence‐specific cleavage of a chimeric linker RNA. The droplet‐encapsulated DNA templates code for the regrowth of new droplets, establishing the proof‐of‐principle for a minimal, genetically encoded cycle of dissolution and regrowth. By directly linking RNA sequence to droplet stability, composition, and life‐cycle dynamics, our work provides a robust platform for engineering evolvable materials and advancing the bottom‐up construction of synthetic cells.

The KANSL1-ARL17A fusion gene generates oncogenic chKANSARL and F-circKA RNAs that synergistically drive lung cancer progression via a novel F-circKA/miR-6860/chKANSARL axis

A novel chimeric RNA RPGR-EEF1A1 enhances autophagy by interaction with the small GTPase RAB37 in a GTP-dependent manner

RNA-RNA interactions tend to be mediated by RNA-binding proteins (RBPs) and are involved in every step of gene regulation. Faithfully mapping RBP-mediated intra- and intermolecular RNA-RNA interactions is a prerequisite for simultaneously inferring noncoding RNA structures and targets. We present a recently developed RIC-seq (RNA in situ conformation sequencing) technology for global profiling of RBP-mediated in situ RNA-RNA interactions. Our approach fixes RBP-mediated RNA-RNA interactions in living cells with formaldehyde for subsequent permeabilization, micrococcal nuclease fragmentation, pCp-biotin labeling, and in situ RNA-RNA proximity ligation. These proximally ligated chimeric RNA fragments are subsequently enriched and converted into pair-end libraries for deep sequencing. Aligning these chimeric fragments to the genome allows the identification of the whole complement of RBP-mediated RNA-RNA interactome in cells and tissue samples. Combining these proximity information and bioinformatic tools enables the unbiased study of RNA structures and targets.

Chimeric RNAs, composed of sequences from different genes, are the least studied category of bacterial RNAs, and their very existence has not been universally recognized so far. The primary aim of this study was to test the hypothesis that unusual RNA chimeras can arise from transcription priming by a specific 8/9-nucleotide RNAs, GCCAAGGC(G). Applying single-molecule sequencing of Escherichia coli cDNA on Oxford Nanopore MinIon platform, we confirmed the expected presence of these 8/9-mers at the 5'-ends of numerous transcripts. Surprisingly, we also discovered thousands of transcripts from diverse genes containing an internal insertion of the GCCAAGGC 8-mer as a replacement of native genomic 3-15 nucleotide sequences. Given that only 1.52 ± 0.47 % of oligonucleotides, containing this 8-mer as a genomic sequence, carry a single-nucleotide substitution within it, the revealed intramolecular “chimeras” likely represent genuine post-transcriptional RNA modifications not compromised by potential artifacts of the experiment. Notably, the substitutions occurred predominantly at sites where the replaced genomic sequences contained GC dinucleotides at both borders. We detected no consensus sequences in the 20-nucleotide flanks near the modification site, implying that signals for intramolecular modifications are located within the replaceable segments. Although detected in over two hundred distinct gene products, most of the chimeric transcripts originated from 16S or 23S rRNAs and were often located within antisense transcripts. The discovered modification has no analogues. While this article discusses its possible biological role, further investigation will require new approaches and fresh conceptual ideas.

Family with sequence similarity 72 (FAM72) is a protein-coding gene family located on chromosome 1 (chr1) in humans, and its functional roles, particularly in cancer, remain incompletely understood. Chimeric messenger RNA (mRNA) generated by intergenic mRNA trans-splicing (CRTS) is a novel phenomenon increasingly recognized for its involvement in cancer biology. It involves the fusion of two separate mRNA transcripts from different genomic loci, resulting in a chimeric mRNA molecule with altered functions. Since aging-related diseases, including various types of cancer, are major threats to our society, we investigated the functional significance of chimeric FAM72 fusion genes and their potential role in cancer cell proliferation with a focus on intergenic mRNA trans-splicing of FAM72 in cancer. We applied biocomputational analyses to identify chimeric FAM72 fusion genes across various cancer tissues. Whole-genome sequencing (WGS) and next-generation sequencing (NGS) of mRNA (RNA-seq) analysis were applied to identify novel chimeric FAM72 fusion genes at the genome (genomic structural variants or SVs) and/or mRNA level (trans-splicing). Our data supported the occurrence of novel chimeric adiponectin receptor-2 (ADIPOR2) :: FAM72B mRNA transcripts primarily produced through an intergenic mRNA trans-splicing event. Afterwards, we set up a breast cancer tissue-specific cell system to analyze the proliferative and migratory efficacy of cancer cells expressing these novel chimeric ADIPOR2::FAM72B mRNA fusion transcripts. Our findings suggest that the novel chimeric ADIPOR2::FAM72B mRNAs, generated by intergenic mRNA trans-splicing, can act as oncogenic drivers and represent promising diagnostic and therapeutic targets in breast cancer.

Molecular mechanisms by which benzo[ghi]perylene promotes cell proliferation and DNA damage via downregulation of the chimeric RNA TVP23C-CDRT4/miR-24–3p axis

Genetic variation at 19q13.3 critically modulates chemical carcinogen‐induced lung carcinogenesis, particularly in mediating the activity of benzo[a]pyrene (B[a]P), a major polycyclic aromatic hydrocarbon (PAH) carcinogen. The adjacent genes ERCC1 and iASPP within this locus respectively coordinate nucleotide excision repair of PAH‐induced DNA damage and suppression of apoptotic pathways. Their synergistic interaction regulates pivotal molecular events during PAH‐driven lung carcinogenesis, ultimately impacting cellular repair, proliferation, and apoptosis. Chimeric RNAs have been increasingly recognized as promising biomarkers and therapeutic targets in cancer. However, the characterization of lung cancer‐specific chimeric RNAs in the context of chemical carcinogenesis remains limited. This study identifies and characterizes ERCC1‐iASPP, a novel chimeric RNA derived from the neighboring genes ERCC1 and iASPP, which exerts tumor‐promoting functions via coding and non‐coding mechanisms. First, the chimeric transcript encodes a previously uncharacterized protein, Ei, which enhances USP45‐mediated deubiquitination of ERCC1, thereby stabilizing ERCC1 protein. Additionally, ERCC1‐iASPP also functions as a (long non‐coding chimeric RNA, lnccRNA): in the cytoplasm, it acts as a (competing endogenous RNA, ceRNA) by sequestering miR‐143‐3p, leading to derepression of CDK1 and PGK1 and subsequent activation of oncogenic pathways, while in the nucleus, ERCC1‐iASPP further promotes transcriptional activation by recruiting STAT4 to the PGK1 promoter. Collectively, these findings establish ERCC1‐iASPP as a bifunctional RNA with both protein‐coding and non‐coding regulatory roles that cooperatively promote B[a]P‐induced lung tumorigenesis. This study highlights ERCC1‐iASPP as a potential diagnostic and therapeutic target in smoking‐related lung cancer.

Messenger RNA (mRNA) is typically produced enzymatically through in vitro transcription using wild-type T7 RNA polymerase; however, this enzyme is also known to generate double-stranded RNA (dsRNA) impurities during transcription. These impurities may evoke an immune response, potentially reducing the therapeutic index of the drug product. Upstream and downstream approaches may be used to mitigate the formation or removal of such dsRNA impurities. However, these processes can be costly, reduce yield, and be challenging to scale for clinical manufacturing. In this work, we engineered a chimeric T7 RNA polymerase by tethering a DNA-binding domain to increase the selectivity of the polymerase for DNA templates and found that it was capable of reducing dsRNA formation. The chimeric T7 RNA polymerase reduced dsRNA levels by three- to four-fold relative to wild-type T7 RNA polymerase with commensurate reduction in immune stimulation in vitro. Additionally, the chimeric T7 RNA polymerase showed improved salt tolerance and was active at NaCl concentrations up to 150 mM, which is otherwise a restrictive condition for wild-type T7 polymerase. These features make this novel enzyme an attractive option for addressing various challenges facing the field of mRNA manufacturing.

Abstract Gliomas are the most prevalent and aggressive primary brain tumors in adults, characterized by rapid progression, therapeutic resistance, and a dismal prognosis. The discovery of targetable fusion genes in cancer has spurred interest in detecting chimeric RNAs for precision treatments. This study identified and validated a novel C19orf47-AKT2 fusion in glioma, including an out-of-frame C19orf47e9-AKT2e2 and an in-frame C19orf47e9-AKT2e3 variant, via RNA sequencing and Sanger sequencing. These chimeric RNAs were undetectable in all five non-tumorous brain tissue samples from epilepsy patients but were present in a significant proportion of glioma samples, indicating tumor specificity. Specifically, they were detected in 88.9% (144/162) of glioma core tissues, higher than that in peritumoral tissues (65.2%, 30/46, p<0.001). Quantitative analysis revealed no significant expression difference between these variants in peritumoral tissues. However, in tumor core tissues, the C19orf47e9-AKT2e2 variant was significantly more abundant (p<0.001). High expression of this variant was associated with poor prognosis in glioblastoma patients (p<0.05). Further analysis revealed that C19orf47-AKT2 chimeras arise from cis-splicing of adjacent genes, involving intergenic pre-mRNA splicing without DNA rearrangement. The in-frame variant is predicted to encode a fusion protein containing domains from both parent genes, and this coding potential was validated by transfecting an expression plasmid into 293T cells. However, no endogenous fusion protein was detected in GBM tissues or patient-derived GBM cells, even after proteasome inhibition, implying that the C19orf47-AKT2 fusion may not function through protein translation. We therefore knocked down the expression of C19orf47-AKT2 chimeras via RNA interference techniques. The subsequent CCK-8 assays revealed that the proliferative capacity of GBM primary cells was significantly inhibited following the down-regulation of these chimeric RNAs. In conclusion, this study identifies novel C19orf47-AKT2 chimeras, generated through cis-splicing, that might function as non-coding RNAs to promote glioblastoma proliferation. These chimeras represent potential prognostic markers and therapeutic targets for gliomas.

The CYP1A1/ chimeric RNA RPL17-C18orf32 axis mediates Benzo[ghi]perylene induced-respiratory toxicity and DNA damage in vitro and in vivo.

IntroductionNon-classical neoantigens at the fusion junctions of chimeric RNAs are tumor- specific with a low risk of autoimmunity and therefore represent ideal targets for personalized vaccines. We present a platform to discover immunogenic neoantigens that drive CD8+ T cell clonotypes from chimeric RNA fusion junctions to promote tumor-reactive T cell expansion and prevent tumor recurrence following immunotherapies.MethodsRNA sequencing data from 15 Lung Adenocarcinoma and 15 Squamous Cell Carcinoma patients (tumor and adjacent normal tissues) were analyzed. The KIF5B [Exon 1-15] | RET [Exon 12- 19] fusion was selected from a patient-derived xenograft (PDX) model based on its established role as an actionable cancer driver in an independent tumor with the same junction. We assessed the affinity of neopeptides from the KIF5B-RET fusion to MHC Class I molecules using in silico tools MHCNuggets and MixMHCPred 2.ResultsHLA-C07:02 showed the highest affinity for 9-mer peptideswith NNDVKEDPK, which emerged as the strongest binder based on HLA-Arena docking and binding energy calculations. Immunogenicity was evaluated by IFNg Enzyme-Linked Immunosorbent Spot (ELISpot) assays using HLA-C07:02- matched Peripheral Blood Mononuclear Cells (PBMCs) from two donors. CD8+ T cells from both donors responded to specific junction peptides. Single-cell 5’gene expression RNA sequencing and T Cell receptor mapping of activated T cells identified 15 TCR clonotypes, five of which had high activation. Key residues in CDR3a and CDR3b are crucial for CD8+ T cell activation. NNDVKEDPK and KEDPKWEFP showed minimal cross-reactivity with the normal tissues.DiscussionThis study demonstrates a robust pipeline for identifying and validating immunogenic neoantigens from chimeric RNAs to design personalized cancer vaccines with high immunogenicity and low cross-reactivity.

This study proposes the implementation of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology for gene therapy targeting genetic mutations in human lymphocytes affected by chronic lymphocytic leukemia (CLL), offering new opportunities for effective treatment of this heterogeneous disease. It focuses on the application of CRISPR–Cas9-mediated targeted sequencing to systematically characterize the biological effects of monoallelic and biallelic TP53 gene lesions, aiming to replace mutant TP53 genes in CLL cells through this technology. CRISPR–Cas9 technology employs a specific enzyme guided by a designed guide RNA (gRNA) to a DNA target. The enzyme first introduces a cut at the target site, and following this cleavage event, it can further disrupt the TP53 gene. The gRNA plays a crucial role by directing the Cas9 protein to the DNA sequence of interest. The gRNA consists of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) sequences, responsible for target recognition and Cas9 binding, respectively. Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA. Although the natural tracrRNA–crRNA mechanism operates efficiently, the use of a single RNA-guided Cas9 system is particularly attractive due to its potential for programmed DNA cleavage and genome editing. Importantly, Cas9 can bind and cleave a target sequence only if it is adjacent to a protospacer adjacent motif. Once the gRNA–Cas9 complex binds to the target DNA, Cas9 induces a double-strand break at the specified site. In conclusion, CRISPR–Cas9 technology represents a powerful genetic engineering tool capable of inserting, deleting, or replacing DNA within an organism’s genome using these “molecular scissors.”

Human papillomavirus (HPV) infection is a primary driver of cervical cancer. Integration of HPV into the human genome causes persistent expression of viral oncogenes E6 and E7, which promote carcinogenesis and disrupt host genomic function. However, the impact of integration on host gene expression remains incompletely understood. We used multimodal RNA sequencing, combining total RNA-seq and Cap Analysis of Gene Expression (CAGE), to clarify virus–host interactions after HPV integration. HPV-derived transcripts were detected in 17 of 20 clinical samples. In most specimens, transcriptional start sites (TSSs) showed predominant early promoter usage, and transcript patterns differed with detectable E4 RNA region. Notably, the high RNA expressions of E4 region and viral-human chimeric RNAs were mutually exclusive. Chimeric RNAs were identified in 13 of 17 samples, revealing 16 viral integration sites (ISs). CAGE data revealed two patterns of TSS upregulation centered on the ISs: a two-sided pattern (43.8%) and a one-sided pattern (31.3%). Total RNA-seq showed upregulation of 12 putative cancer-related genes near ISs, including MAGI1-AS1, HAS3, CASC8, BIRC2, and MMP12. These findings indicate that HPV integration drives transcriptional activation near ISs, enhancing expression of adjacent oncogenes. Our study deepens understanding of HPV-induced carcinogenesis and informs precision medicine strategies for cervical cancer.

Urinary exosomal FAM153C-RPL19 chimeric RNA as a diagnostic and prognostic biomarker for prostate cancer in Chinese patients.

Hepatitis B virus (HBV) is a small DNA virus that establishes chronic infection and drives progressive liver disease and cancer; presenting a global health problem with more than 250 million infections. HBV replicates via an episomal covalently-closed-circular DNA (cccDNA) and integrated viral DNA fragments are linked to carcinogenesis. Current treatments only suppress HBV replication and there is a global initiative to develop genome targeting therapies, including siRNAs, antisense oligonucleotides and epigenetic modifiers specific for HBV cccDNA. However, our knowledge of the cccDNA and integrant transcriptomes is confounded by overlapping viral RNAs. Using targeted long-read sequencing we mapped the HBV transcriptome in liver biopsies from eleven treatment naïve patients. Probe enrichment yielded robust sequencing libraries and identified cccDNA-derived genomic and sub-genomic transcripts, and a repertoire of previously uncharacterised spliced, truncated and chimeric viral RNAs. Assigning viral transcripts to their respective DNA templates revealed differential promoter activity in cccDNA and integrants, with implications for the efficacy of epigenetic modifiers. Integrant-derived transcripts showed vast diversity in the viral-host junctions, posing a challenge for current nucleotide-targeting therapies. cccDNA was a source of genetic polymorphism, with distinct viral lineages present in the surface antigen encoding region, providing an insight into hepadnavirus evolution during chronic infection.