Definition and discovery
Obelisks are a recently identified class of heritable RNA elements associated with microbial communities. They were first described by Zheludev and colleagues in 20241 through a reference-free analysis of large-scale metatranscriptomic datasets, in which candidate RNAs were nominated based on molecular signatures and then filtered to retain contigs lacking evident homology to standard sequence databases.
The authors use the term “obelisk” to refer to these RNA-only agents as a group distinct from known subviral satellites, viroids, and hepatitis delta virus–like agents, while noting that their biological behaviors (including transmission, host impact, and replication mode) remains uncharacterized and cannot yet be assigned based on available data.
Obelisks are defined operationally by three shared properties: (i) ~1 kb circular RNA genome assemblies, (ii) predicted rod-like secondary structures (Figure 1), and (iii) open reading frames encoding a novel protein superfamily termed “Oblins”. Applying this definition, the study identified tens of thousands of related circular RNA assemblies, which resolve into approximately 30,000 operational taxonomic units when clustered at 90% nucleotide identity, indicating that obelisks constitute a widespread and highly diverse group of RNA elements at the sequence level.
Figure 1. Predicted secondary structure of Obelisk-S.s. Modified from Maddamsetti and You (2025)2 on top and zoomed portion below. Obelisks are predicted to adopt a highly elongated, rod-like secondary structure resulting in a molecule that is largely double stranded along most of the genome, with limited number of short internal looks and terminal stem-loops interruptions.
Association with bacterial hosts
In the original study by Zheludev and colleagues1, obelisks were detected in ~50% of queried oral metatranscriptomes and 7% of stool datasets, indicating a high prevalence in at least one oral microbiome dataset collection. This prevalence estimate is derived specifically from publicly available human oral RNA-seq datasets and should not be generalized to all microbiomes.
Beyond prevalence alone, the study establishes a defined host association: a specific lineage, obelisk-S.s, is consistently observed in transcriptomes of Streptococcus sanguinis SK36, a well-characterized oral commensal bacterium.
Extensive analyses of matched nucleic acid preparations found RNA-seq reads mapping to obelisk-S.s but no corresponding signal in DNA sequencing, supporting the conclusion that obelisk-S.s is maintained as an RNA-only element in this host under the experimental conditions tested. In comparative growth experiments, substrains that had apparently lost obelisk-S.s did not show discernible growth defects in replete aerobic culture, consistent with the paper’s conclusion that maintenance is not essential for host viability under standard laboratory growth conditions.
Independent follow up environmental metatranscriptomic analyses have since reported closely related circular RNA elements with obelisk-like features in marine and hot springs ecosystems, further supporting a broad ecological distribution3,4.
Functional status
The biological function of obelisks remains undetermined. The study provides no direct evidence for autonomous replication, does not identify a dedicated polymerase, and explicitly states that transmission mode, host impact, and replication mechanism cannot currently be assigned.
Obelisk genomes are predicted to fold into extended rod-like secondary structures spanning most of the circular genome, reminiscent of classical plant viroids. Comparative and computational analyses indicate strong structural constraint, reflected in conserved base-pairing across divergent sequences, although precise in vivo folding and potential life-cycle–dependent variation remain unknown.
A subset of obelisks contains variant hammerhead type-III self-cleaving ribozyme motifs, detected using an obelisk-specific covariance model. However, catalytic activity was not experimentally tested, and ribozyme function is inferred solely from sequence and structural similarity; additional ribozyme diversity may therefore exist.
Although Oblin ORFs establish protein-coding potential and computational fold predictions are provided, Oblin expression, translation, and biochemical activity have not been experimentally validated. Consequently, any functional role for Oblins remains hypothetical. The coexistence of extensive predicted RNA secondary structure with protein-coding regions raises mechanistic questions about translation, but proposed models such as transient unfolding or host-assisted remodeling remain speculative and should be treated as hypotheses rather than established mechanisms.
Methods for studying Obelisks: detection, validation, and characterization
Obelisks are non-polyadenylated and should be studied using a total RNA-seq stranded workflow such as the NEXTFLEX™ Rapid Directional RNA-seq 2.0 Kit combined with rRNA depletion (for example with the NEXTFLEX RiboNaut™ rRNA Depletion Kit upstream or with NEXTFLEX CRISPR-based depletion downstream).
Alternatively, as in the case of circRNA in eukaryotic cells, samples can be treated with RNase R and then processed with a total RNA-seq workflow. RNase R is a 3’ to 5’ exonuclease commonly used to degrade linear RNAs but spares circular RNAs (including obelisk RNA) due to their closed-loop structure. This enzyme was used in the original study to demonstrate resistance to exonucleolytic degradation. However, the authors emphasize, that RNase R resistance is supportive but not definitive, given that highly structured linear RNAs can also persist.
Functional investigation will depend on tractable host systems, with S. sanguinis SK36 serving as the current benchmark. Systematic curing, reintroduction, and transcriptomic perturbation experiments are proposed by the authors as next steps, alongside biochemical characterization of Oblins and experimental probing of RNA structure.
Conclusion
Obelisks constitute a newly discovered class of viroid-like RNAs found across human-associated and environmental microbiomes. They expand the set of RNA-only genetic elements in nature and may represent a large, previously missed layer of microbiome biology. Supported evidence establishes their existence, circular topology, conserved rod-like secondary structure, broad ecological distribution, and association with specific bacterial hosts, including Streptococcus sanguinis SK36. At the same time, their replication strategy, protein function, and biological impact remain unresolved. Their discovery uncovers a hidden RNA biosphere and offers new insights into RNA evolution, as well as host-microbe interaction5.
By combining RNA sequencing, junction-aware analysis, enzymatic validation, and functional studies, the field is now positioned to move from discovery toward mechanistic understanding of these unusual RNA entities, with the coming years likely to resolve many of the outstanding questions.
References:
- Zheludev, I.N., et al. (2024). Viroid-like colonists of human microbiomes. Cell. 187(23):6521-6536.e18. doi: 10.1016/j.cell.2024.09.033.
- Maddamsetti, R., You, L. (2025). The Abundance of Viroid-Like RNA Obelisk-S.s in Streptococcus sanguinis SK36 May Suffice for Evolutionary Persistence. J Mol Evol. 93(3):370-378. doi: 10.1007/s00239-025-10250-y.
- López-Simón, J., et al. (2025). Viroid-like "obelisk" agents are widespread in the ocean and exceed the abundance of RNA viruses in the prokaryotic fraction. ISME J. 19(1):wraf033. doi: 10.1093/ismejo/wraf033.
- Urayama, S.I., et al. (2025). Identification of Obelisk-like covalently closed circular RNA replicon in hot springs by double-stranded RNA sequencing and expansion of the diversity of the Obelisk superfamily. bioRxiv [Preprint]. Sep 3:2025.09.03.673927. doi: 10.1101/2025.09.03.673927.
- Li, L., et al. (2025). A novel viroid-like RNA element “Obelisks”: a major breakthrough in the RNA World. Mol Biomed. 6, 47. Doi:10.1186/s43556-025-00290-7
