Circular DNA & RNA: Biochemistry, Molecular Biology, and Synthetic Modifications, Nucleic Acid Technology
Luke McLaughlin, Biotech Digital Marketer, Business Developer and Life Science Content Creator
Circular DNA and RNA molecules, distinguished from their linear counterparts by their covalently closed loop structures, have become pivotal in the fields of molecular biology and biotechnology. These molecules, including plasmids, circular RNA (circRNA), and synthetic constructs, exhibit unique stability and regulatory characteristics, making them valuable for various applications. This review delves into the biochemistry and molecular biology of circular DNA and RNA, explores the range of synthetic modifications possible, and discusses their implications for therapeutic and biotechnological applications.
Circular DNA and RNA are naturally occurring and synthetically engineered molecules characterized by their covalently closed, continuous loop structures. This configuration confers resistance to exonucleases, contributing to their enhanced stability compared to linear nucleic acids. In nature, circular DNA is commonly found in plasmids and the mitochondrial DNA of many organisms, while circRNAs, typically formed by back-splicing events, play critical roles in gene expression regulation.
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.
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.
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.