Circular RNAs

The Secret Circular World of Genetic Regulation

From biological curiosity to medical frontier

The Hidden RNA Universe - Circular RNAs Emerge from the Shadows

In the fascinating world of molecular biology, where linear DNA molecules and their RNA transcripts have long been the center of attention, a surprising architectural alternative has emerged—circular RNAs. These mysterious circular molecules, once dismissed as mere splicing errors, have now revolutionized our understanding of genetic regulation and opened new frontiers in medical science. Unlike their linear counterparts, circular RNAs form covalently closed loops without the traditional beginning and end, making them remarkably stable and capable of unique biological functions ¹ .

Stable Structure

Covalently closed loops without 5' cap or 3' tail make circRNAs resistant to degradation by exonucleases.

Medical Potential

From cancer progression to neurodegenerative disorders, circRNAs offer promising avenues for novel therapies and diagnostic biomarkers ³ ⁹ .

Circular RNA Basics: The Accidental Discovery That Revolutionized RNA Biology

What Are Circular RNAs?

Circular RNAs (circRNAs) are a unique class of RNA molecules characterized by their covalently closed circular structure. Unlike linear RNAs, they lack the traditional 5' cap and 3' poly(A) tail, which makes them exceptionally resistant to degradation by cellular exonucleases. This structural stability allows them to persist in cells for much longer than most linear RNAs, making them ideal candidates for cellular regulation and potential therapeutic applications ¹ ⁶ .

The Journey From Junk to Jewel

The transformation in our understanding of circular RNAs represents one of the most dramatic shifts in modern molecular biology. Initially dismissed as transcriptional noise, circular RNAs are now recognized as important regulators of gene expression with tissue-specific distribution and evolutionary conservation across species ¹ .

Key Discoveries in circRNA Research

1976

First suspicion of circular RNAs when scientists discovered plant viroids consisted of single-stranded circular RNA molecules ³ .

1990

First observation of circular RNAs in mammalian cells, specifically in a tumor suppressor gene called DCC ³ .

Early 2010s

Technological advances revealed the abundance and functional significance of circular RNAs, transforming our understanding ⁶ .

How Cells Create Perfect RNA Circles: The Art of Molecular Origami

The Backsplicing Mechanism

The formation of circular RNAs is a remarkable example of molecular origami that defies traditional splicing rules. While linear RNAs are produced through canonical splicing (joining 5' donor to 3' acceptor sites), circular RNAs are generated through a process called backsplicing, where a downstream 5' splice site joins with an upstream 3' splice site ¹ ⁶ .

Intron-pairing-driven cyclization

Complementary sequences within introns flanking the circularized exons base-pair with each other ³ .

RBP-driven cyclization

Specific proteins such as muscleblind (MBL) and Quaking (QKI) bind to intronic sequences and promote circularization ⁶ ⁹ .

Lariat-driven cyclization

Exon skipping events can generate lariat structures containing exons, which then undergo internal splicing ³ .

Types of Circular RNAs

Type	Composition	Cellular Location	Primary Functions
exonic circRNAs (ecircRNAs)	Exons only	Cytoplasm	miRNA sponging, protein binding
exon-intron circRNAs (EIciRNAs)	Both exons and introns	Nucleus	Regulating transcription
circular intronic RNAs (ciRNAs)	Introns only	Nucleus	Modulating transcription
tRNA intronic circRNAs (tricRNAs)	tRNA introns	Cytoplasm	Unknown regulatory functions

Table 1: Types of Circular RNAs and Their Characteristics ³ ⁶

Molecular Multitaskers in the Cell: The Diverse Functions of Circular RNAs

miRNA Sponges: The Molecular Decoys

One of the most well-established functions of circular RNAs is their role as microRNA sponges. These circular molecules contain multiple binding sites for specific microRNAs, effectively sequestering them and preventing them from targeting their natural mRNA targets. This "sponging" activity allows circular RNAs to indirectly regulate gene expression by modulating microRNA activity ¹ ⁹ .

The most famous example of this sponge function is CDR1as (also known as ciRS-7), which contains more than 70 conserved binding sites for miR-7 and is highly expressed in human and mouse brains ³ ⁶ .

Protein Interactors

Circular RNAs can bind to proteins and alter their function, serve as scaffolds for assembling multiprotein complexes, and regulate transcription through interactions with RNA polymerase II ³ .

Translation Potential

Some circular RNAs can be translated into proteins through internal ribosome entry sites (IRES) or modifications such as N6-methyladenosine (m6A) that promote cap-independent translation ³ ⁶ .

Circular RNAs in Cancer, Neurodegeneration and Beyond: The Disease Connection

Circular RNAs in Cancer

The role of circular RNAs in cancer represents one of the most actively researched areas in the field. Generally, circular RNAs tend to be downregulated in tumor tissue compared to normal tissue, which may result from errors in the back-splice machinery in malignant tissues, degradation by deregulated miRNAs, or reduced stability due to increased cell proliferation ¹ .

circRNA	Cancer Type	Function	Mechanism
circPVT1	Head and neck squamous cell carcinoma	Oncogenic	Sponges miR-497-5p, regulates proliferation genes
circHIPK3	Nasopharyngeal carcinoma	Oncogenic	Promotes cancer progression
circ-FBXW7	Glioblastoma	Tumor suppressor	Encodes FBXW7-185aa protein that inhibits c-Myc
circ-ITCH	Various cancers	Tumor suppressor	Sponges miRNAs that target tumor suppressors

Table 2: Examples of Circular RNAs Involved in Human Cancers ³ ⁹

Neurological Disorders

circRNAs are highly abundant in neuronal tissues and have been linked to Alzheimer's disease ³ .

Cardiovascular Diseases

Multiple circRNAs have been associated with myocardial infarction, cardiac hypertrophy, and fibrosis .

Wound Healing

CircGLIS3(2) increases in skin fibroblasts after injury and promotes tissue rebuilding ⁷ .

Chemical Circularization: Engineering RNA Circles for Therapy

The Experimental Breakthrough

One of the most significant technical advances in circular RNA research comes from a landmark study published in Nature Communications in 2025, which developed a novel method for chemically circularizing in vitro transcribed RNAs of various lengths (35-4000 nucleotides) with circularization efficiencies reaching up to 60% ⁸ .

This innovative approach, called chemical circularization, leverages a 5' ethylenediamine modification and a periodate-oxidized 3' end to drive intramolecular reductive amination. This process forms a morpholine-derived inter-nucleotide linkage that creates a stable circular structure ⁸ .

Step-by-Step Methodology

Primer design and synthesis: Researchers designed two types of AG dinucleotides equipped with ethylenediamine linkers of different lengths ⁸ .
In vitro transcription: The team generated in vitro transcribed (IVT) pre-circRNA that is 5' functionalized with ethylenediamine ⁸ .
Chemical circularization: The functionalized pre-circRNA was circularized through a one-pot periodate oxidation and reductive amination (PORA) reaction ⁸ .
Purification and validation: The researchers developed effective separation methods to isolate chem-circRNAs from their linear precursors ⁸ .

Comparison of Circularization Methods

Method	Efficiency	Limitations	Advantages
Chemical circularization	Up to 60%	Requires specialized primers	Sequence flexibility, modification compatibility
Enzymatic ligation	Variable	Sequence constraints, purification challenges	Biological compatibility
Ribozymatic circularization	Variable	Incompatible with modified bases	No enzyme requirement
Group I/II intron circularization	Variable	Specific sequence requirements	High fidelity in vivo circularization

Table 3: Comparison of Circularization Methods ⁸

Essential Tools for Circular RNA Investigation

The study of circular RNAs requires specialized reagents and methodologies that have been developed and refined over recent years. Here we highlight key research reagent solutions that enable circular RNA discovery and characterization:

Sequencing & Detection

circRNA-enriched RNA sequencing kits
Backsplice junction detection probes
Single-cell circRNA sequencing reagents

Functional Study Tools

circRNA-specific knockdown reagents
Circularization reporter systems
Synthetic circRNA delivery systems

Analytical Reagents

RNase R enzyme
circRNA quantification assays
circRNA-protein interaction kits

The Circular RNA Revolution: From Biological Curiosity to Medical Frontier

The journey of circular RNAs from dismissed artifacts to key regulatory molecules represents one of the most exciting developments in molecular biology in recent decades. These stable, abundant, and versatile molecules have been revealed as important players in virtually all aspects of cellular function and in numerous disease processes ¹ ⁹ .

The circular RNA revolution reminds us that scientific humility is essential—what we once dismissed as cellular debris often turns out to be biological treasure.

Future Directions

Novel circRNA-based diagnostics that leverage the stability and tissue-specific expression of circular RNAs for early disease detection
circRNA therapeutics that exploit the natural functions of circular RNAs as miRNA sponges and protein decoys
circRNA expression platforms that utilize engineered circular RNAs for durable protein production
Artificial intelligence-driven discovery that accelerates our identification and functional characterization of circular RNAs ² ⁴