Circular DNA & RNA

Understanding the Role of Circular DNA and RNA in Gene Therapy, Synthetic Biology, and Next-Gen Biotech Tools

In recent years, circular nucleic acids have emerged as key players in molecular biology, synthetic biology, and therapeutic innovation. Unlike their linear counterparts, circular DNA and RNA molecules are defined by their covalently closed, looped structures, which afford them unique biochemical properties and functional advantages. These circular forms, both naturally occurring and synthetically engineered, include well-known entities such as plasmids and circular RNAs (circRNAs), as well as designer constructs developed for specific biotechnological and medical purposes. Their structural continuity protects them from exonucleolytic degradation, endowing them with enhanced stability, prolonged half-lives, and superior resistance to cellular enzymes.

Circular DNA molecules are ubiquitous in nature, particularly within prokaryotic organisms, where plasmids play vital roles in horizontal gene transfer, antibiotic resistance, and metabolic adaptation. In eukaryotic systems, extrachromosomal circular DNA (eccDNA) and mitochondrial DNA exist in circular configurations and are increasingly recognized for their involvement in genome plasticity, gene regulation, and disease progression. These molecules have been instrumental in the development of recombinant DNA technologies, gene therapy vectors, and DNA-based vaccines, demonstrating their versatility and value in both fundamental and applied biosciences.

Similarly, circular RNA represents a rapidly expanding frontier in transcriptomics and functional genomics. Discovered more than two decades ago but long dismissed as splicing artifacts, circRNAs are now recognized as stable and abundant RNA species generated primarily through back-splicing events. They are known to regulate gene expression by acting as microRNA (miRNA) sponges, modulating transcription, interacting with RNA-binding proteins, and even translating into functional peptides in some contexts. Their cell- and tissue-specific expression patterns, stability in biofluids, and regulatory potential make them promising candidates for biomarker discovery and therapeutic targeting.

This review aims to provide a comprehensive overview of circular DNA and RNA molecules, with a focus on their biochemical and molecular characteristics, formation mechanisms, and biological functions. It also explores the latest advances in synthetic modifications—both chemical and structural—that enhance the stability, functionality, and therapeutic potential of these nucleic acid structures. Finally, we discuss the growing range of applications across gene therapy, vaccine development, molecular diagnostics, and synthetic biology, highlighting the transformative impact of circular nucleic acids in modern biotechnology and medicine.

Biochemistry and Molecular Biology of Circular DNA

Structure and Formation

Circular DNA is primarily found in plasmids—small DNA molecules within bacteria and sometimes in eukaryotic cells. These molecules replicate independently of chromosomal DNA and are essential for gene transfer across species, antibiotic resistance, and metabolic functions. Circularization of DNA involves the formation of covalent bonds that stabilize the structure, thereby protecting genetic information.

Biological Functions

Circular DNA molecules are crucial for horizontal gene transfer among prokaryotes, facilitating rapid adaptation and evolution. In eukaryotes, extrachromosomal circular DNA can influence gene expression and genomic stability, impacting cancer progression and cellular aging.

Biochemistry and Molecular Biology of Circular RNA

Structure and Formation

Circular RNA (circRNA) is typically formed by a non-canonical splicing process called back-splicing, where a downstream splice donor is joined to an upstream splice acceptor. This process can involve intronic lariats that escape debranching, leading to exon-containing circRNAs.

Biological Functions

CircRNAs are highly abundant in the eukaryotic transcriptome and perform various roles, including acting as miRNA sponges, regulating transcription, and influencing RNA-binding protein (RBP) sequestration. These functions are integral in cellular processes such as differentiation, proliferation, and apoptosis.

Synthetic Modifications for Therapeutic and Biotechnological Applications

Chemical Modifications

Chemical modifications in synthetic circular RNA and DNA can enhance their stability and binding affinity. Common modifications include methylation of the ribose sugar, incorporation of phosphorothioate backbones, and attachment of lipid or polyethylene glycol (PEG) groups to improve cellular uptake and circulation time.

Therapeutic Applications

Circular DNAs, especially plasmids, are employed in gene therapy and vaccines, offering a stable vector for gene delivery. Circular RNAs, due to their stability and capacity to sequester miRNAs, are being explored as novel therapeutic agents in cancer therapy, cardiovascular diseases, and neurodegenerative disorders.

Biotechnological Applications

In biotechnology, circular DNA is utilized in the production of recombinant proteins, vaccine development, and synthetic biology. Circular RNAs can serve as versatile tools in molecular diagnostics and as potential biomarkers due to their cell-type-specific expression patterns and stability.

Delivery Systems

Delivery systems are not just a footnote in nucleic acid therapeutics; they are the central bottleneck. A beautiful strand of mRNA, siRNA, or a circular RNA is useless if it can't survive the bloodstream, cross cell membranes, avoid immune surveillance, and reach the right cell type and subcellular compartment. Let's dive deep into the molecular engineering behind each major delivery vehicle, with a focus on their physical chemistry, cellular trafficking, and targeting strategies.

1. Lipid Nanoparticles (LNPs)

LNPs are the most clinically validated platform, especially for mRNA delivery (e.g., SARS-CoV-2 vaccines). They form amphiphilic vesicles, typically 60–150 nm in diameter, optimized for endosomal escape and nucleic acid protection.

Key Components and Their Functions:

1. Ionizable Cationic Lipids (e.g., DLin-MC3-DMA):
2. Helper Lipids (e.g., DSPC, DOPE):
3. Cholesterol:
4. PEGylated Lipids:

Mechanism of Action:

• Formulation: Microfluidic mixing rapidly combines ethanol-dissolved lipids with aqueous nucleic acid, driving self-assembly via electrostatic and hydrophobic interactions.
• Uptake: Endocytosis (mostly clathrin-mediated).
• Endosomal Escape: Triggered by protonation of ionizable lipids, promoting membrane fusion and release of cargo into the cytosol.
• Degradation/Excretion: LNPs are ultimately degraded or cleared hepatically.

2. Exosomes / Extracellular Vesicles (EVs)

Biogenesis & Structure:

• Size: 30–150 nm (exosomes), derived from multivesicular bodies (MVBs).
• Composition: Lipid bilayer containing tetraspanins (CD9, CD63, CD81), integrins, heat shock proteins, and endogenous RNA/protein cargo.

Therapeutic Engineering:

• Loading strategies:
• Targeting: Surface engineering using:
• Advantages:
• Challenges:

3. Aptamer-Mediated Delivery

Definition:

• Aptamers are short, single-stranded DNA or RNA oligonucleotides (20–100 nt) that fold into 3D structures capable of binding specific targets with high affinity (KD in the nM–pM range).

Delivery Mechanism:

• Aptamers are conjugated to:

Design Considerations:

• Target selection: Cell-surface receptors (e.g., PSMA, EGFR)
• Stability: Aptamers are prone to degradation; chemically modified bases (e.g., 2'-fluoro, 2'-O-methyl) and PEGylation are used for stabilization.
• Endosomal escape is typically poor, so co-delivery with endosomolytic agents (e.g., fusogenic peptides) may be required.

4. Polymeric Nanoparticles

Types:

1. PLGA (poly(lactic-co-glycolic acid)):
2. PEI (polyethyleneimine):

Key Features:

• Surface Modifications:
• Charge Ratio (N/P ratio):
• Limitations:

Emerging Hybrid Systems

1. Lipid–polymer hybrid nanoparticles:
2. Inorganic carriers (e.g., gold NPs, mesoporous silica):

Circularization Techniques in vitro

Circular RNA (circRNA) synthesis in vitro is a nontrivial bottleneck in both research and therapeutic development. Efficient and clean circularization is critical to avoid linear RNA contaminants (which can be immunogenic or unstable) and to ensure proper translation, if the circRNA encodes protein.

The enzymatic ligation approach to circRNA production primarily involves ATP-dependent ligases such as T4 RNA ligase 1 (Rnl1) and T4 RNA ligase 2 (Rnl2). Rnl1 preferentially ligates single-stranded RNA molecules that present a 5′-phosphate and a 3′-hydroxyl, making it suitable for circularizing linear RNA with compatible termini. In practice, linear RNAs synthesized by in vitro transcription often require enzymatic 5′ phosphorylation using T4 polynucleotide kinase prior to ligation. Since Rnl1 is most efficient when the 5′ and 3′ ends of the RNA are brought into spatial proximity, circularization efficiency is enhanced by introducing complementary DNA or RNA splints, which hybridize to the RNA termini and coax them into a ligation-competent conformation. Rnl2 is generally more selective for double-stranded substrates or nicks within RNA/DNA duplexes, but truncated versions (e.g., Rnl2 truncated K227Q) are frequently used in splint-mediated circularization because of their high fidelity and lower off-target ligation. However, enzymatic ligation efficiency is often hampered by RNA secondary structures, especially for longer transcripts (>1 kb), which can obstruct enzyme access to the termini. Moreover, this method is prone to the generation of byproducts, including re-ligated linear transcripts, dimers or concatamers (through intermolecular ligation), and nicked or partially ligated species, especially in the absence of precise stoichiometric and folding control.

An alternative and often more efficient approach is ribozyme-mediated circularization, particularly via the group I intron-based permuted intron–exon (PIE) system. This method takes advantage of the self-splicing activity of group I introns, originally characterized in Tetrahymena thermophila and other organisms. The PIE design rearranges the natural intron-exon architecture to place the coding or functional sequence between two halves of the group I intron, creating a transcript that undergoes autocatalytic splicing upon transcription. The ribozyme catalyzes two transesterification reactions: first, a guanosine cofactor attacks the 5′ splice site, releasing the 5′ intron half; then, the free 3′-OH of the upstream exon (which corresponds to the end of the circular RNA) attacks the 3′ splice site, ligating the two flanking exons and releasing the circularized RNA. This process produces a covalently closed circRNA and a linear excised intron. Ribozyme-based methods offer relatively high efficiency under optimized conditions but are sensitive to folding kinetics, Mg²⁺ concentrations, and sequence context. Misfolding or mis-splicing can lead to aberrant RNA species, including uncleaved precursors, partially processed intermediates, or mis-spliced variants. The correct design of exon–intron boundaries and careful in vitro transcription and folding protocols are essential for efficient circularization using this method.

Beyond biological enzymes, chemical circularization techniques provide a more modular but often less biologically faithful method for covalently joining RNA ends. One of the most classical strategies involves carbodiimide-mediated activation of the 5′ phosphate—commonly using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 1,1′-carbonyldiimidazole (CDI)—to form a reactive phosphorimidazolide intermediate. The 3′-OH of the RNA then performs a nucleophilic attack, resulting in phosphodiester bond formation. This approach can proceed in the absence of proteins, but it requires careful pH control, the presence of stabilizing cations like Mg²⁺ or Zn²⁺, and a splint oligonucleotide to juxtapose the reactive ends. Chemical ligation is often inefficient for long RNAs due to hydrolysis of intermediates, RNA backbone cleavage, or low regioselectivity, especially when the ends are not well-aligned or accessible. A separate class of chemical circularization uses bioorthogonal reactions such as copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), also known as “click chemistry.” In this method, the 5′ and 3′ ends of the RNA are functionalized with alkyne and azide groups, respectively, and reacted in the presence of Cu(I) and a ligand. The resulting triazole linkage is robust and stable but non-native, raising concerns about biocompatibility and immunogenicity in therapeutic applications.

A central challenge across all circularization methods is the efficient removal of linear RNA contaminants, which can persist due to incomplete ligation, failed splicing, or re-ligation events. These linear RNAs are problematic because they are sensitive to exonucleases, can trigger innate immune responses via RIG-I or MDA5 sensing, and may result in off-target translation products. To purify circRNAs, several post-processing steps are commonly used. RNase R digestion is the most standard biochemical method—this 3′-to-5′ exoribonuclease efficiently degrades linear RNA species while sparing circRNAs, which lack accessible ends. However, RNase R treatment is not perfect; it can sometimes digest certain structured circRNAs or leave behind nicked linear fragments. Additional purification strategies include denaturing PAGE, which exploits the altered electrophoretic mobility of circular RNA (due to its topological constraints), as well as high-performance liquid chromatography (HPLC) and capillary electrophoresis, which offer higher resolution separation for analytical and preparative purposes. Finally, specific junction-spanning probes can be used in RT-qPCR or northern blotting to selectively detect and quantify circRNA species, distinguishing them from linear precursors.

For therapeutic or translational circRNAs, another layer of complexity arises: circularization must be performed in such a way that preserves critical functional elements, including internal ribosome entry sites (IRES), RNA modifications such as m⁶A, and uninterrupted open reading frames (ORFs). Misplacing the circular junction can disrupt translation efficiency or protein coding capacity. Furthermore, the choice of circularization method may constrain the placement of these elements or introduce unwanted junctional sequences. Thus, the trade-off between efficiency, biological fidelity, and scalability must be carefully balanced when selecting a circularization strategy for synthetic circRNAs intended for research or therapeutic deployment.

Synthetic Modifications of Circular DNA and RNA

Chemical Modifications

Chemical modifications are crucial for enhancing the stability and functional capabilities of circular DNA and RNA in therapeutic contexts. These modifications can help protect these molecules from degradation by nucleases, increase their half-life in biological systems, and improve their affinity for target molecules. Some common modifications include:

Methylation: Addition of methyl groups (CH3) to the ribose sugar in RNA (2'-O-methylation) or to the bases in DNA and RNA can protect nucleic acids from degradation and improve binding specificity.

Phosphorothioate Backbones: Replacing a non-bridging oxygen in the phosphate backbone of nucleic acids with sulfur can enhance resistance to nucleases and promote binding to proteins, aiding in delivery and function.

Peptide Nucleic Acids (PNAs): These are synthetic polymers that mimic DNA and RNA structures but have a peptide backbone. PNAs are not recognized by proteases or nucleases, making them extremely stable.

Conjugation with Lipid or Polyethylene Glycol (PEG): Conjugation can enhance cellular uptake and improve solubility and circulation time in the bloodstream. For instance, lipid nanoparticles are commonly used for the delivery of mRNA vaccines.

Common Nucleobase Modifications

Nucleobase modifications in circRNAs aim to alter their stability, binding specificity, and immune reactivity. Here are some of the critical nucleobase modifications:

• Pseudouridine (Ψ): This modification, where uridine is converted to pseudouridine, is known to enhance the stability of RNA molecules and reduce immune recognition. Pseudouridine is particularly effective in circRNAs because it can improve translational efficiency without compromising the structural integrity of the RNA.
• 5-methylcytosine (m5C): Methylation of cytosine bases in circRNAs can influence their interaction with proteins and other cellular components. This modification is crucial for fine-tuning the function of circRNAs, particularly in gene expression regulation and RNA-protein interactions.
• N6-methyladenosine (m6A): This is one of the most prevalent modifications in eukaryotic mRNA and is also applicable to circRNAs. m6A modifications can promote the export of circRNAs from the nucleus to the cytoplasm and enhance their translation or degradation, depending on the context, thereby modulating gene expression post-transcriptionally.

Common Backbone Modifications

Modifications to the phosphate backbone of circRNAs are primarily designed to enhance their nuclease resistance and improve pharmacokinetic properties:

• Phosphorothioate (PS) Bonds: Replacing a non-bridging oxygen in the phosphate backbone with sulfur results in the formation of phosphorothioate bonds. This modification significantly enhances the resistance of circRNAs to nucleases, increasing their stability in biological fluids. PS-modified circRNAs are particularly valuable in therapeutic applications where RNA stability is crucial, such as in antisense oligonucleotides and siRNA therapies.
• 2'-O-Methylation: In this modification, the 2' hydroxyl group of the ribose sugar is methylated. This not only protects circRNAs from enzymatic degradation but also reduces their immunogenicity, making them more suitable for therapeutic applications. 2'-O-methylation is crucial for increasing the circRNAs' half-life in the circulatory system, thus enhancing their efficacy as therapeutic agents.
• Locked Nucleic Acids (LNAs): Incorporation of LNAs, which involve a methylene bridge connecting the 2'-oxygen with the 4'-carbon of the ribose ring, locks the ribose in the ideal 3'-endo conformation for Watson-Crick binding. This enhances hybridization affinity to complementary RNA or DNA and increases resistance to nuclease degradation. LNA-modified circRNAs are used in therapeutic settings where high-affinity binding and excellent biostability are required.

Additional Nucleobase Modifications

• Inosine (I): Inosine is an adenosine analog where the amino group is replaced with a keto group. This modification can increase base-pairing flexibility and can impact RNA splicing and editing processes. In circRNAs, inosine can modulate interactions with RNA-binding proteins and potentially alter the circRNA's functionality.
• Queuosine (Q): This is a modified guanine base that is found in tRNA and is also applicable to other RNA types, including circRNAs. Queuosine modification can affect RNA folding and stability, which might influence the interactions of circRNAs with other cellular components.
• 4-Thiouridine (s4U): Thiouridine involves replacing the oxygen in the uridine base with sulfur. This modification is sensitive to UV light, providing a tool for cross-linking RNA to interacting proteins, which can be particularly useful in studying the interaction dynamics of circRNAs within the cell.
• Hydroxymethylcytosine (hm5C): This is a derivative of 5-methylcytosine where an additional hydroxymethyl group is attached. This modification is known to affect the epigenetic regulation of DNA, and when applied to RNA, it could potentially influence RNA stability and interactions with proteins.
• Base Editing: Direct chemical editing of RNA bases through deamination, which converts adenosine to inosine (A-to-I editing) or cytosine to uridine (C-to-U editing), can be utilized to alter the coding potential of circRNAs or modify their interaction with microRNAs and other RNAs.
• Azobenzene-modified Nucleobases: Incorporating azobenzene groups into nucleobases can make the RNA responsive to light, allowing for the reversible control of RNA structure and function with light exposure. This can be particularly useful in developing RNA-based switches and sensors.
• Selex-Nucleotide Modifications: These are modifications selected through systematic evolution of ligands by exponential enrichment (SELEX) to produce nucleotide analogs with high specificity and binding affinity for particular targets. This can be used to create circRNAs with precise targeting capabilities for specific RNA sequences or proteins.
• Clickable Nucleosides: Incorporation of alkyne or azide groups into nucleosides allows for subsequent click chemistry reactions, enabling the post-synthetic conjugation of various functional groups, labels, or even drug molecules to RNA. This is particularly useful for designing circRNA-based therapeutics where targeting or delivery to specific cellular compartments is needed.
• Isothermal Nucleic Acid Modifications: These modifications include structures that exhibit high stability at elevated temperatures or in harsh chemical environments, enhancing the practical utility of circRNAs in industrial biotechnology applications, such as bio-catalysis and synthetic biology.
• Triple Helix Forming Oligonucleotides (TFOs): These are not direct modifications of the RNA itself but involve the use of oligonucleotides that can bind to the major groove of double-stranded RNA, forming a triple helix. This can be used to control gene expression or inhibit the function of specific RNA molecules.
• Caged Nucleobases: These are nucleobases modified with photolabile protecting groups that can be removed by light exposure. This allows for the precise control of RNA activity through light, enabling studies on RNA function in real-time within living cells.
• Base Analogs Sensing Modifications: Insertion of fluorescent or other reporting analogs can be used to monitor RNA interactions and dynamics within biological systems, providing insights into RNA folding, decay, and protein interactions.

Additional Backbone Modifications

• Boronophosphate (BH3): Substitution of a non-bridging oxygen in the phosphate backbone with a boron group can alter the charge and reactivity of the backbone. Boronophosphate modifications can enhance nuclease resistance and affect the overall biophysical properties of circRNAs.
• Methylphosphonate: This modification involves replacing one of the non-bridging oxygens in the phosphate group with a methyl group, resulting in a neutral backbone. Methylphosphonate-modified RNAs show enhanced resistance to nucleases and decreased affinity for water, which can be useful in therapeutic applications where nuclease stability is crucial.
• Peptide Linkages: Incorporating peptide bonds into the RNA backbone can drastically change the properties of the molecule. These 'peptide nucleic acids' (PNAs) are not recognized by cellular enzymes, making them resistant to degradation and effective in hybridization-based applications.
• Threose Nucleic Acid (TNA): TNA is an artificial genetic polymer where the ribose sugar in RNA is replaced with threose sugar. TNAs are resistant to nuclease degradation and can bind to RNA and DNA, making them useful for therapeutic and diagnostic applications.
• Glycol Nucleic Acids (GNA): Similar to TNA, GNA involves replacing the sugar backbone with a glycol unit. GNAs are highly stable and form very stable duplexes with complementary DNA or RNA strands, useful for molecular recognition applications.
• Xeno Nucleic Acids (XNAs): This is a broad category of synthetic nucleic acid analogs that include several types of modified sugar backbones, such as those found in TNA and GNA. XNAs can have diverse properties depending on their sugar backbone modifications, and they are designed to improve stability, affinity, and specificity in binding to natural nucleic acids.
• Sulfur Replacement in Backbone (Phosphorodithioate): In this modification, both non-bridging oxygens in the phosphate backbone are replaced with sulfur. This significantly enhances nuclease resistance more than the single sulfur replacement seen in phosphorothioates and can also impact the electrostatic properties of the RNA.
• Morpholino Phosphorodiamidate: This backbone modification, commonly seen in morpholino antisense oligonucleotides, involves a morpholine ring replacing the ribose and phosphorodiamidate linkages instead of phosphate. This configuration results in increased nuclease resistance and decreased immune stimulation, useful for therapeutic applications where circRNA stability is critical.
• Amide-Linked Nucleic Acids (ALNAs): Here, the phosphodiester backbone is replaced with an amide linkage. ALNAs exhibit high biostability and improved binding affinity to complementary RNA or DNA, making them excellent candidates for therapeutic gene regulation.
• Bridged Nucleic Acids (BNAs): BNAs, including LNAs (locked nucleic acids), contain a bridge that locks the ribose in an ideal conformation for binding. BNAs enhance hybridization properties and are used in applications where high specificity and strong binding are necessary, such as in vivo gene silencing.
• Polyethylene Glycol (PEG) Modification: Attaching PEG molecules to the RNA backbone can increase solubility and reduce immunogenicity, which is particularly useful for therapeutic applications where longer circulation times and lower immune responses are desired.
• Chiral Backbone Modifications: These involve altering the stereochemistry of the RNA backbone, potentially impacting the way RNA interacts with proteins and other biomolecules, which can be tailored for specific therapeutic interactions.
• Sulfur and Selenium Modifications: Replacing oxygen in the phosphate backbone with sulfur or selenium can enhance nuclease resistance and alter the electronic properties of the backbone, impacting RNA's interaction with proteins and other nucleic acids.

Enhanced Modifications for Specific Applications

• Fluorination: Introducing fluorine atoms, particularly at the 2' position of ribose (2'-fluoro), can greatly stabilize RNA by enhancing hydrophobicity and resistance to enzymatic degradation. This modification is particularly valuable in therapeutic applications where circRNA stability in the bloodstream or within cellular environments is necessary.
• Ribose Ring Modifications: Modifying the ribose sugar itself, such as through the introduction of bridging groups that 'lock' the ribose in a favorable conformation (as in Locked Nucleic Acids), can enhance binding specificity and stability against degradation.

Exotic Modifications

• Photo-cleavable Nucleosides: Incorporation of nucleosides that can be cleaved by light allows for the spatial and temporal control of RNA functions. This is particularly useful in research and therapeutic applications where controlled activation or deactivation of RNA functions is needed.
• Heavy Atom-modified Nucleosides: Inserting heavy atoms like iodine or bromine into nucleosides can enhance the x-ray contrast of RNA, useful for structural studies using crystallography or other imaging techniques.
• Metal-chelating Nucleosides: These are designed to bind specific metal ions, facilitating the use of circRNAs in metal-ion sensing or in catalysis processes where metals play a critical role.

Therapeutic Applications

Gene Therapy: Circular DNA such as plasmids are used as vectors to deliver therapeutic genes into cells. Their circular structure ensures that they are stable and can persist in host cells without integration into the host genome, reducing the risk of mutagenesis.

Cancer Therapy: Circular RNAs can act as miRNA sponges to sequester miRNAs that promote cancer cell proliferation. By binding to these miRNAs, circRNAs can inhibit their function and thereby suppress tumor growth.

Vaccine Development: Circular DNA is employed in DNA vaccines, where it is used to express antigenic proteins that stimulate an immune response. The stability of circular DNA ensures effective expression of the vaccine antigen.

Circular DNA beyond plasmids (e.g., eccDNA, telomeric circles)

1. Extrachromosomal Circular DNA (eccDNA)

eccDNAs are covalently closed circular DNA molecules that originate from chromosomal DNA but exist outside the chromosomes, typically in the nucleus or cytoplasm. They can vary widely in size—from hundreds of base pairs (microDNAs) to megabase-scale fragments (double minutes).

Biogenesis

eccDNAs are generally thought to arise from aberrant repair of DNA double-strand breaks (DSBs) or from replication and recombination errors. Key pathways implicated in their formation include:

• Non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) for small eccDNAs.
• Homologous recombination (HR), especially in repetitive regions, for large eccDNAs.
• R-loop-induced DNA damage and transcriptional stress may also promote eccDNA formation near active genes.

Features

• Typically lack centromeres, rendering them non-mitotically stable unless they are exceptionally large or anchored to nuclear structures.
• Gene-rich eccDNAs may contain enhancers, promoters, or entire oncogenes, allowing them to drive transcription independently of chromosomal position.

2. MicroDNAs

MicroDNAs are a subset of eccDNAs typically between 100–400 base pairs in size. They are found in both normal and cancerous cells and originate from non-repetitive, GC-rich genomic regions, often near transcription start sites, 5′ untranslated regions (UTRs), or exons.

Biogenesis

MicroDNAs are believed to result from:

• DNA polymerase slippage, leading to excision of short DNA loops.
• Mismatch repair-associated excision, particularly during replication.
• Aberrant nucleosome positioning and chromatin remodeling.

Functional Consequences

Though they do not typically carry entire genes, microDNAs can:

• Serve as sources of regulatory RNAs (e.g., small interfering RNAs or piRNAs) via transcription and processing.
• Act as immune-stimulatory agents, activating cytosolic DNA sensors like cGAS-STING, particularly when exported from the nucleus.
• Function as substrates for recombination, contributing to genomic instability.

3. Double Minutes (DMs)

Double minutes are large, acentric, extrachromosomal circular DNA elements, typically hundreds of kilobases to megabases in length. They are frequently observed in cancer cells, particularly in glioblastoma, neuroblastoma, and ovarian cancers.

Molecular Characteristics

• DMs lack centromeres and telomeres, making them mitotically unstable. However, during replication, they can be unevenly segregated, leading to copy number heterogeneity across daughter cells.
• Often carry amplified oncogenes such as MYC, EGFR, MDM2, or CDK4.
• Frequently include cis-regulatory elements, such as super-enhancers, enabling high-level oncogene expression.

Functional Relevance

• Because of their high copy number and lack of chromosomal regulation, DMs drive oncogene overexpression more effectively than standard chromosomal amplification.
• Their non-Mendelian inheritance creates tumor cell heterogeneity, promoting clonal evolution and therapeutic resistance.
• DMs can integrate into chromosomal DNA (a process called "episome reintegration") or be lost entirely, providing dynamic oncogene dosage modulation.

4. Telomeric Circles (t-circles and c-circles)

Telomeric circles are circular DNA molecules composed entirely or predominantly of telomeric repeat sequences (e.g., TTAGGG repeats in humans). These are particularly relevant in cancer cells that employ the Alternative Lengthening of Telomeres (ALT) pathway instead of telomerase.

Types

• T-circles: Double-stranded closed DNA molecules formed from excised telomeric repeats.
• C-circles: Covalently closed partially single-stranded telomeric DNA species. These are amplified in ALT-positive tumors and serve as biomarkers of ALT activity.

Biogenesis

Telomeric circles are believed to form through:

• Recombination between telomeric repeats (particularly via t-loop excision).
• Replication fork collapse at telomeric DNA during ALT.
• Exonuclease-mediated resection and ligation.

Functional Roles

• Act as donor templates for telomere extension by homologous recombination, a key feature of ALT.
• May be byproducts of t-loop resolution, a process regulated by SLX1-SLX4-MUS81 endonucleases.
• Their presence correlates with genomic instability, particularly at subtelomeric regions.

Relevance in Cancer and Genomic Instability

Circular DNA species are increasingly recognized as drivers of cancer biology, particularly through:

1. Oncogene amplification: eccDNAs and DMs allow high expression levels and rapid modulation of gene dosage.
2. Intratumoral heterogeneity: Due to their unequal segregation and independent replication, they create cell-to-cell differences in oncogene content.
3. Therapeutic resistance: eccDNAs can be lost or gained under selective pressure, allowing dynamic adaptation to chemotherapy or targeted agents.
4. Immune signaling: eccDNAs and microDNAs in the cytosol can trigger innate immune pathways (e.g., cGAS-STING), which may either enhance anti-tumor immunity or promote chronic inflammation and tumor progression.

Detection and Analysis

Several high-resolution techniques are employed to characterize circular DNA species:

• Circle-Seq / eccDNA-seq: Uses exonuclease digestion of linear DNA followed by rolling circle amplification and sequencing.
• Electron microscopy: Direct visualization of circular DNA morphology.
• Fluorescence in situ hybridization (FISH): Used to localize DMs and eccDNAs carrying specific oncogenes.
• C-circle assays: Quantify C-circles specific to ALT+ telomeres.

Biotechnological Applications

Synthetic Biology: Circular DNA is extensively used to build synthetic biological circuits and pathways. For example, they can be engineered to contain promoter sequences and regulatory elements that control the production of proteins or metabolic pathways in microbial factories.

Molecular Diagnostics: Circular RNAs, due to their cell- and tissue-specific expression and extraordinary stability, are excellent candidates for biomarkers in diagnosing diseases such as cancer or neurodegenerative disorders.

Research Tools: Modified circular RNAs can be used as molecular sponges or decoys in research, allowing scientists to study the function of specific proteins or regulatory RNAs by sequestering them and observing the resulting phenotypic changes.

The exploration of circular DNA and RNA in this review underscores their pivotal roles in molecular biology, biotechnology, and therapeutic interventions. These covalently closed loop structures offer unique stability and regulatory characteristics, making them invaluable assets in various applications.

From a biochemical and molecular biology standpoint, the understanding of circular DNA's presence in plasmids and its significance in horizontal gene transfer among prokaryotes, as well as circRNAs' diverse functions in eukaryotic gene expression regulation, highlights the complexity and versatility of these molecules in cellular processes.

Moreover, the array of synthetic modifications discussed demonstrates the potential to tailor circular DNA and RNA for specific therapeutic and biotechnological purposes. Chemical modifications, nucleobase alterations, and backbone modifications offer strategies to enhance stability, binding affinity, and resistance to degradation, crucial for their efficacy in therapeutic interventions and biotechnological applications.

Therapeutically, circular DNA serves as stable vectors in gene therapy and vaccine development, while circular RNAs exhibit promise as novel therapeutic agents, particularly in cancer therapy and cardiovascular diseases, owing to their stability and regulatory roles in gene expression.

In biotechnology, circular DNA finds utility in synthetic biology for building biological circuits and pathways, while circular RNAs emerge as valuable molecular tools for molecular diagnostics and as research aids due to their specific expression patterns and stability.

In essence, the comprehensive understanding of circular DNA and RNA, coupled with synthetic modifications, opens avenues for innovation and advancement in various fields, promising impactful contributions to healthcare, biotechnology, and scientific research. As such, continued exploration and refinement of these circular nucleic acids hold immense potential for addressing diverse challenges and driving progress in molecular biology and biotechnology.