Synthetic RNAs in Focus: Exploring the Functional Terrain of 100–500mers

Harnessing RNA Versatility for Therapeutic, Diagnostic, and Biotechnological Innovation

Synthetic RNA molecules spanning 100–500 nucleotides (nt) have emerged as exceptionally versatile tools at the intersection of molecular biology, biotechnology, and medicine. This size range occupies a unique sweet spot long enough to encode complex secondary structures and functional domains, yet compact enough for efficient synthesis, modification, and delivery. These mid-length RNAs have enabled breakthroughs across a wide spectrum of applications, from programmable therapeutics and smart diagnostics to regulatory circuits in synthetic biology. Unlike shorter oligonucleotides or full-length mRNAs, RNAs in this intermediate size class offer a powerful balance between molecular stability, structural complexity, and design flexibility. As a result, they have become central to cutting-edge research in gene editing, targeted drug delivery, RNA-based sensing, and immunotherapy, making them indispensable tools in both fundamental science and translational innovation.

Small-fragment mRNA vaccines

Small-fragment mRNA vaccines (100–500 nucleotides) precisely encode defined antigenic epitopes or modular vaccine components. These synthetic constructs typically encode peptides of 30–150 amino acids, selected via computational and experimental epitope mapping (e.g., IEDB). Codon optimization is employed to enhance translational efficiency and stability, removing cryptic splice sites and secondary structures (hairpins), as predicted by RNAfold or MFold algorithms. Additionally, synthetic mRNAs incorporate optimized 5'-cap structures (m7GpppN or Cap-1) and tailored 3'-UTRs coupled to poly(A) tails (50–150 adenine residues) for increased translation efficiency and stability against nuclease degradation.

Manufacturing methods for these small mRNAs include solid-phase chemical synthesis (for ~100–200 nt) or in vitro transcription (IVT) with bacteriophage polymerases (T7, SP6) for longer constructs. During IVT, modified nucleoside triphosphates like pseudouridine or N1-methylpseudouridine are often introduced to minimize innate immune activation. Purification via high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE), followed by quality assurance through mass spectrometry (LC-MS) and capillary electrophoresis, ensures purity, sequence fidelity, and consistency.

Short mRNA vaccine fragments focus immune responses precisely on defined epitopes, stimulating potent cellular (CD8+ cytotoxic and CD4+ helper T cells) and humoral immunity. The targeted design of these constructs significantly reduces off-target immunogenicity and associated inflammatory or autoimmune risks. Specific applications include vaccines for infectious diseases, such as influenza and SARS-CoV-2, where conserved epitopes provide broad protection, and cancer vaccines encoding patient-specific neoantigens to elicit robust tumor-specific immune responses with reduced systemic toxicity.

Despite their potential, challenges remain, notably in efficient intracellular delivery, requiring sophisticated lipid nanoparticle (LNP) or polymer-based formulations. Comprehensive immunogenic validation through methods such as ELISpot, cytokine release assays, and T-cell receptor sequencing is essential. Future innovations are anticipated in RNA stabilization and delivery technologies, such as novel lipid-polymer conjugates and targeted nanoparticles, as well as multivalent constructs integrating immunostimulatory adjuvants. As this technology matures, small-fragment mRNA vaccines will increasingly represent essential tools for personalized immunotherapy and precision vaccinology.

(CRISPR)

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system relies critically on synthetic single guide RNAs (sgRNAs), typically 100–200 nucleotides long. Each sgRNA comprises a spacer region (20–25 nt) for target specificity through complementary DNA binding and a scaffold region (~80–150 nt) essential for stable binding to Cas nucleases like Cas9 or Cas12a. Structurally, the scaffold contains conserved RNA motifs, including the repeat:anti-repeat duplex, tetraloops (GAAA, UUCG), stem-loops, and nexus structures, crucial for Cas-sgRNA complex stability and efficient DNA cleavage.

Engineering sgRNAs involves careful bioinformatic spacer sequence optimization using tools such as CRISPOR and CHOPCHOP, balancing on-target specificity and reduced off-target editing, typically targeting GC content between 40–60%. Scaffold engineering, incorporating structural mutations or chemical modifications, enhances nuclease stability, reduces immune detection, and boosts enzymatic efficiency. Variants like extended scaffolds or chemically modified sgRNAs with phosphorothioate backbones or 2'-modified nucleosides (2'-O-methyl, 2’-fluoro) significantly improve stability and functionality.

Production methods include precise solid-phase chemical synthesis for shorter sgRNAs (≤150 nt) or in vitro transcription (IVT) with bacteriophage RNA polymerases (T7, SP6) for constructs up to ~500 nt, incorporating modified nucleoside triphosphates for stability and reduced immune activation. Rigorous purification methods, such as PAGE or chromatography, ensure high purity and fidelity. Delivery approaches for sgRNAs include transient ribonucleoprotein (RNP) complexes, viral vectors (lentivirus, AAV), lipid nanoparticles (LNPs), and electroporation, each optimized to enhance cellular uptake, stability, and precise nuclear localization.

Applications span therapeutic genome editing targeting monogenic diseases, high-throughput functional genomics screens using sgRNA libraries, and agricultural biotechnology for crop improvement and microbial metabolic engineering. Challenges remain, including reducing off-target genomic edits, optimizing delivery efficiency, and minimizing innate immune responses. Future developments involve advanced scaffold engineering for greater specificity, novel computational-experimental design integration, and next-generation delivery platforms, positioning CRISPR sgRNAs as pivotal tools in precision medicine and biotechnology innovation.

RNA aptamers

RNA aptamers, typically ranging from 100 to 500 nucleotides, are single-stranded nucleic acid molecules engineered through Systematic Evolution of Ligands by Exponential enrichment (SELEX). SELEX iteratively selects aptamers exhibiting high-affinity, specific binding from vast randomized RNA libraries (up to 10¹⁵ distinct sequences). The selection process includes binding, partitioning, amplification, and sequencing steps, enabling the isolation of RNA molecules that form stable three-dimensional structures such as hairpins, loops, pseudoknots, and G-quadruplexes essential for precise ligand recognition.

Structurally, RNA aptamers fold into intricate secondary and tertiary conformations determined by nucleotide sequence and composition. Aptamer-target interactions primarily involve hydrogen bonds, electrostatic forces, hydrophobic effects, and shape complementarity. Chemical modifications, including 2'-fluoro, 2'-O-methyl substitutions, phosphorothioate linkages, or locked nucleic acids (LNAs), enhance aptamer stability against nuclease degradation, increase binding affinity, and extend functional half-life in biological environments, critical for therapeutic and diagnostic applications.

RNA aptamers have diverse applications in diagnostics and biosensors, capitalizing on their exceptional target specificity and rapid binding kinetics. Aptamers are routinely integrated into assays for pathogen detection, cancer biomarkers, and environmental toxin sensing, outperforming conventional antibodies in stability, reproducibility, and cost-effectiveness. In therapeutics, aptamers function as potent antagonists or agonists, exemplified by FDA-approved Pegaptanib (Macugen) for age-related macular degeneration, demonstrating their clinical potential in targeted molecular therapy.

Despite their versatility, challenges persist in aptamer development, including achieving sustained bioavailability, efficient cellular internalization, and minimizing off-target effects. Advances in aptamer conjugation strategies (e.g., PEGylation, lipidation, nanoparticles) and improved computational modeling for structure prediction are addressing these limitations. Future directions involve developing multiplexed and bispecific aptamers, as well as integrating them into innovative biosensing platforms and targeted drug delivery systems, expanding their role as powerful molecular recognition tools in biotechnology and precision medicine.

Circular RNAs (circRNAs)

Circular RNAs (circRNAs), typically comprising sequences ranging from approximately 100 to several hundred nucleotides, are covalently closed single-stranded RNA molecules generated primarily through a non-canonical splicing event termed back-splicing. In back-splicing, a downstream splice donor site ligates to an upstream acceptor site, resulting in a continuous RNA loop devoid of free 5’ and 3’ ends. This closed-loop structure confers extraordinary stability against exonucleolytic degradation compared to linear RNAs, thereby extending circRNA half-life and persistence in cellular and extracellular environments.

Structurally, circRNAs frequently originate from protein-coding gene loci, comprising one or more exons (exonic circRNAs) or intronic sequences (intronic circRNAs). Their biogenesis involves intricate regulatory mechanisms, influenced by cis-acting elements such as intronic complementary sequences (ICS), inverted Alu repeats, and trans-acting factors including RNA-binding proteins (RBPs) like Quaking (QKI), Muscleblind (MBL), and heterogeneous nuclear ribonucleoproteins (hnRNPs). Computational tools like CIRI, circBase, and find_circ facilitate genome-wide circRNA identification and validation through RNA sequencing coupled with specialized algorithms that detect unique back-spliced junction reads.

CircRNAs possess diverse biological functions predominantly through their roles as competitive endogenous RNAs (ceRNAs), interacting directly with microRNAs (miRNAs), RNA-binding proteins, or translational machinery. Acting as miRNA sponges, circRNAs regulate gene expression by sequestering specific miRNAs, preventing their binding to mRNA targets, and modulating downstream signaling pathways relevant to cellular proliferation, differentiation, and disease progression. Additionally, select circRNAs possess internal ribosome entry sites (IRES) or N6-methyladenosine (m^6A) modifications, enabling translation into small peptides, highlighting their versatility beyond non-coding RNA paradigms.

Given their robust stability, tissue specificity, and dynamic expression profiles, circRNAs are increasingly recognized as valuable biomarkers and therapeutic targets. They have shown promise in diagnostics, particularly in oncology, cardiovascular diseases, and neurodegenerative disorders, detectable in biofluids such as blood, saliva, and cerebrospinal fluid. Therapeutically, engineered synthetic circRNAs, including circRNA vaccines or circRNA-based gene delivery systems, leverage their structural stability for prolonged expression and reduced immunogenicity. Current research advances focus on developing optimized delivery platforms (e.g., lipid nanoparticles), enhancing circRNA translational efficiency, and leveraging their regulatory networks to create novel therapeutic interventions across diverse clinical settings.

RNA switches and ribozymes

RNA switches and ribozymes are functional RNA molecules that act as molecular regulators or catalysts, often within the size range of 100–500 nucleotides. These RNAs can dynamically change conformation or mediate biochemical reactions in response to specific intracellular or extracellular signals. RNA switches also known as riboswitches when naturally occurring undergo structural rearrangements upon binding to a ligand (e.g., small metabolite or ion), leading to downstream regulation of gene expression, typically at the transcriptional or translational level. In contrast, ribozymes are catalytic RNAs capable of site-specific cleavage, ligation, or splicing of RNA molecules without protein assistance, serving essential roles in RNA processing and gene regulation.

Natural ribozymes, such as the hammerhead, hairpin, HDV (hepatitis delta virus), and group I/II introns, catalyze reactions like RNA cleavage or self-splicing through well-defined three-dimensional folds that position functional groups (often divalent metal ions like Mg²⁺) for catalysis. In synthetic biology, engineered ribozymes have been developed for controllable mRNA degradation or activation by inserting them into untranslated regions (UTRs) of transcripts. Their activity can be fine-tuned by rational design or directed evolution, allowing RNA-based circuits to be responsive to molecular cues. These constructs are often used in synthetic gene regulation platforms or to create logic-gated expression systems in therapeutic applications.

RNA switches are often modularly designed and include aptamer domains that recognize ligands (e.g., theophylline, SAM, FMN, thiamine pyrophosphate), coupled to expression platforms that regulate gene output. Ligand binding induces conformational changes that either expose or occlude ribosome binding sites (RBS) or splice sites, thereby modulating translation or splicing. Artificial RNA switches, built via SELEX-derived aptamers, have been used to control mRNA translation in response to synthetic small molecules, enabling precise, reversible, and tunable gene expression in prokaryotic and eukaryotic systems. More advanced versions integrate these RNA devices into CRISPR systems or mRNA therapeutics for ligand-controlled activity.

In biotechnology and therapeutic development, RNA switches and ribozymes are being explored for applications such as smart gene therapies, RNA logic gates, biosensors, and conditional mRNA vaccines. Their programmability, compact size, and minimal reliance on protein cofactors make them attractive for RNA-based therapeutics where spatiotemporal control is crucial. However, challenges include ensuring ligand specificity, avoiding off-target effects, and maintaining proper folding and function in vivo. Ongoing advances in RNA structure prediction, high-throughput screening, and RNA delivery methods continue to expand the utility of RNA switches and ribozymes in synthetic biology, gene therapy, and diagnostic platforms.

Splice-switching RNAs (SSOs)

Splice-switching RNAs (SSOs) are short, synthetic antisense oligonucleotides designed to modulate pre-mRNA splicing by binding to specific sequences within introns or exons. Typically 15–30 nucleotides in length, but often part of longer constructs (up to 100–500 nt when embedded in vector systems), SSOs do not degrade their RNA targets; instead, they sterically block access of the spliceosome to canonical splice sites or splicing enhancers/silencers. This redirection of the splicing machinery results in exon inclusion, exon skipping, or intron retention, offering a powerful mechanism for altering gene expression post-transcriptionally without modifying the genome.

Mechanistically, SSOs work by targeting cis-regulatory splicing elements, such as exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), and their intronic counterparts. Binding of SSOs to these motifs interferes with the recruitment of spliceosomal components (e.g., U1 snRNP, SF2/ASF), shifting the splicing outcome. For example, in Duchenne muscular dystrophy (DMD), SSOs are used to skip mutated exons in the DMD gene, restoring the reading frame and enabling production of a truncated but functional dystrophin protein. In spinal muscular atrophy (SMA), the FDA-approved drug nusinersen enhances inclusion of exon 7 in the SMN2 transcript, compensating for the loss of function in SMN1.

From a chemical standpoint, SSOs are heavily modified to improve in vivo stability, affinity, and target specificity. Common modifications include phosphorothioate backbones (to resist nuclease degradation), 2′-O-methyl, 2′-O-methoxyethyl (MOE), and locked nucleic acids (LNAs) to enhance binding affinity and reduce immunogenicity. When incorporated into AAV vectors or self-splicing circular RNA constructs, longer SSOs (~100–500 nt) can be expressed intracellularly for sustained activity, particularly valuable in gene therapy settings requiring long-term modulation of splicing.

Therapeutically, SSOs have entered clinical use and are being evaluated for a range of disorders beyond DMD and SMA, including certain cancers, beta-thalassemia, and cystic fibrosis. The ability to selectively modify splicing patterns enables correction of disease-causing mutations, isoform switching, or even induction of nonsense-mediated decay (NMD) for gene silencing. Key challenges include achieving efficient tissue-specific delivery (especially to muscle, brain, or liver), avoiding off-target effects, and ensuring consistent splicing outcomes across patient populations. With advancements in delivery platforms (e.g., peptide-conjugates, lipid nanoparticles) and high-throughput screening technologies, splice-switching RNAs are poised to become essential tools in precision RNA therapeutics.

MicroRNA (miRNA) mimics and decoys

MicroRNA (miRNA) mimics and decoys are synthetic RNA-based tools used to modulate gene expression by either enhancing or inhibiting endogenous miRNA activity. miRNAs are ~22-nucleotide non-coding RNAs that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3′ untranslated regions (UTRs) of target mRNAs, leading to translational repression or mRNA degradation. miRNA mimics are synthetic double-stranded RNAs designed to restore or enhance the activity of a specific miRNA that is downregulated in disease. Conversely, miRNA decoys (also known as antagomiRs, miRNA sponges, or competitive inhibitors) are single- or multi-site RNA molecules that sequester endogenous miRNAs, preventing them from binding to their natural mRNA targets.

miRNA mimics typically consist of a guide strand that mimics the endogenous miRNA sequence and a passenger strand designed to promote loading of the guide into the RNA-induced silencing complex (RISC). These synthetic RNAs are chemically stabilized with modifications such as 2’-O-methyl, 2’-fluoro, and phosphorothioate backbones to enhance nuclease resistance and improve cellular uptake. Once loaded into RISC, the guide strand can repress target mRNAs in the same way as native miRNAs, making mimics useful in diseases where tumor-suppressive miRNAs are lost (e.g., miR-34a in cancer). Therapeutic miRNA mimics, such as MRX34, have been explored in clinical trials for cancer, though immune-related toxicities remain a challenge.

miRNA decoys act by competitively binding one or more target miRNAs, thus derepressing their downstream mRNA targets. These decoys can take several forms: synthetic antisense oligonucleotides (e.g., LNA-antimiRs), circular RNAs (circRNA-based sponges), or engineered transcripts with multiple tandem miRNA-binding sites. For instance, miRNA sponges are often designed with bulged binding sites to prevent cleavage by Argonaute2 while maintaining high-affinity binding. Decoys are commonly used in both basic research and therapeutic development to inhibit oncomiRs like miR-21, miR-155, or miR-221, thereby restoring expression of tumor suppressor genes.

The therapeutic utility of miRNA mimics and decoys spans oncology, cardiovascular disease, viral infections, and neurological disorders. However, key challenges include delivery to specific tissues or cell types, off-target effects, and immune activation. Delivery strategies include lipid nanoparticles (LNPs), galactose-targeted conjugates (GalNAc) for liver targeting, and exosome-based systems. Innovations in RNA chemical engineering and nanoparticle-based delivery are driving the clinical translation of miRNA-based therapies. As understanding of miRNA biology deepens, synthetic mimics and decoys are increasingly viewed as potent and programmable tools for post-transcriptional gene regulation and precision medicine.

Small nuclear RNAs (snRNAs)

Small nuclear RNAs (snRNAs) are short non-coding RNA molecules, typically 100–300 nucleotides long, that play essential roles in pre-mRNA splicing as integral components of the spliceosome. The core snRNAs U1, U2, U4, U5, and U6 associate with specific proteins to form small nuclear ribonucleoproteins (snRNPs). These snRNPs recognize conserved sequences at exon-intron boundaries and catalyze the removal of introns from pre-mRNA transcripts through a two-step transesterification reaction. snRNAs contain conserved Sm-binding sites and structured domains (e.g., stem-loops, kink-turns) critical for both snRNP assembly and spliceosomal catalysis.

Synthetic snRNA constructs are engineered for experimental and therapeutic purposes to modify or study splicing. For example, modified U7 snRNAs have been repurposed to deliver antisense sequences that modulate splicing of specific pre-mRNAs, such as skipping mutated exons in Duchenne muscular dystrophy (DMD) or correcting exon inclusion in spinal muscular atrophy (SMA). These synthetic constructs often incorporate antisense elements within the snRNA scaffold while preserving key secondary structures needed for proper nuclear localization, RNP assembly, and interaction with spliceosomal proteins.

For therapeutic delivery, engineered snRNAs are typically expressed from pol III or pol II promoters in plasmids or viral vectors (commonly AAVs) to ensure efficient nuclear transcription and long-term expression in target tissues. Modifications to snRNA sequences can include the addition of specific antisense motifs, Sm-binding site enhancements, or stabilizing structural loops. Functional snRNP formation requires proper interaction with Sm or LSm protein cores, and in some cases, engineered constructs include tags or motifs to direct snRNA localization or facilitate snRNP biogenesis in non-native systems.

Applications of snRNA/snRNP constructs include therapeutic splice correction, trans-splicing, and functional dissection of splicing elements in pre-mRNA. In addition to neuromuscular disorders, these tools are being explored in cancer, inherited metabolic diseases, and viral infections where aberrant splicing plays a pathogenic role. Challenges include ensuring efficient nuclear import, avoiding immune responses to vectorized snRNAs, and achieving isoform-specific effects without disrupting global splicing. With advances in vector engineering, antisense design, and understanding of splicing regulation, synthetic snRNA/snRNP constructs are becoming increasingly valuable in both mechanistic RNA biology and RNA-based therapeutics.

Reporter RNAs

Reporter RNAs are synthetic or engineered messenger RNAs designed to express easily detectable proteins such as luciferases, fluorescent proteins (e.g., GFP, mCherry), or enzyme tags for monitoring gene expression, RNA stability, translation efficiency, or cellular localization. Typically ranging from 100 to 500 nucleotides for minimal constructs, reporter RNAs include a coding region for the reporter protein, often preceded by a 5' untranslated region (UTR) and followed by a 3' UTR and poly(A) tail, enabling efficient translation and proper mRNA stability. They serve as powerful tools in both research and clinical settings to trace cellular processes in real-time with high sensitivity.

In experimental systems, reporter RNA constructs are used to study RNA biology, including ribosome recruitment, mRNA localization, splicing, and post-transcriptional regulation. By inserting regulatory elements (e.g., IRES, miRNA binding sites, RNA switches) into UTRs of the reporter construct, researchers can analyze how specific RNA motifs or binding proteins affect RNA translation or degradation. Bicistronic or dual-reporter systems (e.g., Renilla/Firefly luciferase) allow normalization and quantitative comparisons of translational control or promoter activity in various biological contexts.

Reporter RNAs are also widely applied in mRNA vaccine and drug delivery research as surrogates for therapeutic mRNAs. Short reporter constructs are used in lipid nanoparticle (LNP) formulations to assess cellular uptake, delivery efficiency, and in vivo protein translation without the need for therapeutic payloads. For example, luciferase reporter mRNAs can be formulated and injected into animal models, enabling rapid quantification of protein expression using bioluminescence imaging. Similarly, fluorescent protein reporters allow live-cell tracking and single-cell analysis using flow cytometry or microscopy.

In therapeutics, synthetic reporter RNAs can be employed for functional screening, vector optimization, and bioavailability studies. They enable non-invasive monitoring of tissue-specific delivery and expression kinetics, which is critical for optimizing delivery vehicles in mRNA therapeutics and gene editing platforms. As tools for regulatory element characterization and delivery validation, reporter RNAs are essential for translational RNA research. Future innovations may involve self-reporting therapeutic mRNAs that co-express a reporter peptide or use split-reporter systems activated only upon successful delivery and translation, further refining RNA-based diagnostics and precision medicine platforms.

RNA barcodes and indexes

RNA barcodes and indexes are short, synthetic RNA sequences typically 10–100 nucleotides in length engineered to uniquely tag individual molecules, cells, or experimental conditions. These sequences are embedded in larger RNA constructs or transcribed independently and are not translated into proteins. Instead, they serve as unique molecular identifiers (UMIs) or barcodes that allow the tracking, quantification, and deconvolution of complex biological mixtures. RNA barcoding is essential in high-throughput experiments such as single-cell RNA sequencing (scRNA-seq), CRISPR screens, and synthetic circuit tracking, where thousands to millions of elements must be simultaneously identified and analyzed.

Structurally, RNA barcodes are composed of randomized or pre-defined nucleotide sequences inserted into non-coding regions such as UTRs, introns, or dedicated barcode modules within transcripts. These regions are designed to be transcriptionally neutral and minimally disruptive to RNA structure or function. In scRNA-seq, for example, unique cell barcodes and UMIs are attached to each RNA molecule during reverse transcription via barcoded primers, enabling digital counting of transcripts and differentiation of thousands of cells within a single reaction. Barcode fidelity and error correction are supported by Levenshtein distance design and redundancy schemes to distinguish true biological variation from sequencing noise.

In pooled CRISPR screens, RNA barcodes are linked to sgRNA expression cassettes to track which perturbation each cell receives. Similarly, synthetic biology applications use RNA barcodes to label and trace the behavior of engineered RNA devices or gene circuits in microbial or mammalian systems. RNA indexing can also facilitate combinatorial screening, where mixtures of regulatory elements, gene variants, or drug responses are multiplexed and demultiplexed using specific RNA barcodes read by next-generation sequencing. This enables massive scalability and precise mapping of genotype-phenotype relationships in a single experiment.

The success of RNA barcode systems depends on efficient design, integration, and reliable readout. Challenges include barcode sequence cross-talk, secondary structure formation, and barcode dropout due to transcriptional silencing or degradation. Advances in high-throughput oligo synthesis, barcode error correction algorithms, and integration into droplet- and microfluidics-based platforms have greatly enhanced the robustness of RNA barcoding technologies. Moving forward, innovations such as dynamic barcodes, time-stamped RNAs, and RNA barcoding in vivo will further enable precise lineage tracing, cell fate mapping, and high-resolution systems biology studies.

Self-replicating RNA molecules

Self-replicating RNA molecules, often derived from viral genomes (such as alphaviruses, flaviviruses, or nodaviruses), carry built-in replication machinery typically encoding RNA-dependent RNA polymerase (RdRP) that enables autonomous amplification within host cells. These replicon RNAs usually span several kilobases; however, non‑replicating fragment libraries focus on modular, truncated versions that exclude replication genes yet retain elements enabling subgenomic amplification. Such designs allow packaging of distinct payloads within subgenomic regions that are amplified only when co-delivered with helper replication proteins, offering controlled expression without full autonomous replication.

Structurally, these constructs maintain cis-acting replication elements such as the 5' and 3' untranslated region (UTR) sequences and internal promoter elements necessary for RdRP-mediated recognition and subgenomic transcription. For instance, alphavirus-based systems incorporate the conserved 3' terminal sequence, a subgenomic promoter upstream of a payload region (~100–1000 nt), and terminator signals. The payload fragments themselves are synthetic RNAs designed as libraries (~100–500 nt) to express variant peptides, antigens, regulatory domains, or molecular barcodes. Payloads are delimited by unique flap sequences or insulators to prevent recombination and cross-reactivity in pooled formats.

Library generation typically involves synthesizing diverse oligonucleotide pools via microarray-based synthesis holding thousands to millions of distinct fragment sequences, followed by amplification and cloning into the subgenomic region of a replicon backbone. In non-replicating fragment (NRF) libraries, helper RNAs encoding viral RdRP proteins (but lacking capsid or envelope genes) are co-transfected in trans, enabling payload amplification without generating infectious particles. This split-replicon approach offers tight biosafety controls and modularity. After co-delivery into cells, payloads are amplified by replicase activity, allowing screening for expression, protein function, or phenotypes using high-throughput readouts such as sequencing, FACS, or reporter activation.

Applications of NRF libraries span vaccine discovery, epitope mapping, functional peptide screening, and synthetic evolution. In vaccinology, self-replicating fragment libraries allow rapid mapping of immunodominant epitopes by expressing variant antigen fragments at high levels in antigen-presenting cells. In protein engineering, libraries of enzymatic domains can be functionally selected in situ. Key challenges include ensuring uniform library representation post-amplification, avoiding recombination-induced chimeras among library members, and carefully tuning helper:payload ratios. Future innovations involve integrating RNA-based barcodes for lineage tracking, developing self-amplifying RNA-vectored systems with regulated replicase expression, and combining non-replicating fragment libraries with lipid-nanoparticle delivery for in vivo screens and personalized antigen discovery.

ASO–RNA hybrids

ASO–RNA hybrids represent a novel class of engineered oligonucleotide therapeutics that combine the gene-silencing specificity of antisense oligonucleotides (ASOs) with the structural and functional versatility of RNA scaffolds. These hybrid constructs are typically designed by annealing a chemically stabilized ASO (usually 15–25 nucleotides) to a complementary region within a longer synthetic RNA molecule (~100–500 nt), forming a stable duplex that can be further functionalized. The RNA portion can include structural motifs, aptamers, or barcodes for delivery targeting or regulatory control, while the ASO moiety mediates cleavage or modulation of a target RNA through RNase H recruitment or steric hindrance.

Mechanistically, ASO–RNA hybrids enhance targeted delivery and uptake by incorporating ligand elements into the RNA scaffold such as aptamers for cell-specific receptors (e.g., AS1411 for nucleolin, transferrin receptor aptamers, or GalNAc for liver targeting). These ligands guide the hybrid molecule to desired cell types, facilitating receptor-mediated endocytosis. Once internalized, endosomal escape is either assisted by structural features or through nanoparticle encapsulation. Upon cytoplasmic release, the ASO component binds its target mRNA and induces gene knockdown via RNase H-mediated degradation or splicing modulation, depending on the backbone and chemical modifications (e.g., phosphorothioate, 2’-MOE, LNA).

From a design perspective, ASO–RNA hybrids offer modularity: the RNA scaffold provides a flexible platform for including secondary structures, targeting domains, or even internal ribosome entry sites (IRES) to allow cotranslation of therapeutic payloads. This approach enables the co-delivery of ASOs and functional RNAs, such as miRNA decoys, guide RNAs, or non-coding regulatory elements, in a single construct. In some designs, the RNA portion can act as a decoy or sponge, while the ASO targets a separate mRNA, allowing dual-functionality within a single molecule. High-throughput screening using barcoded hybrid libraries allows for rapid optimization of delivery efficiency, tissue specificity, and gene-silencing efficacy.

Therapeutically, ASO–RNA hybrids are being explored for diseases requiring cell-type-specific gene silencing, such as cancers, neurological disorders, and metabolic diseases. Compared to naked ASOs, the hybrid format improves in vivo pharmacokinetics, enhances cellular uptake, and reduces off-target effects by incorporating programmable RNA features. Challenges include ensuring hybrid stability in serum, optimizing endosomal escape, and balancing duplex formation with functional release of the ASO. As RNA engineering and delivery technologies advance, ASO–RNA hybrids represent a powerful platform for precision-targeted gene modulation with enhanced delivery control and multifunctional potential.

Long RNA probes

Long RNA probes are synthetic or in vitro–transcribed RNA molecules typically ranging from 300 to several thousand nucleotides in length, designed to hybridize to complementary RNA or DNA targets with high specificity. Unlike short oligonucleotide probes, long RNA probes allow multi-region binding, increasing hybridization stability and sensitivity, especially for applications involving rare or structured targets. These probes are often labeled with fluorescent dyes, biotin, or digoxigenin for detection and are widely used in Northern blotting, in situ hybridization (ISH), RNA pull-down assays, and targeted RNA capture protocols.

The design of long RNA probes requires careful consideration of sequence composition, secondary structure, and labeling strategy. Probes are usually generated by in vitro transcription using T7, SP6, or T3 RNA polymerases from DNA templates flanked by promoter sequences. Antisense RNA probes (complementary to the target) are the most common format, ensuring specific binding to endogenous mRNAs or non-coding RNAs. To enhance signal strength and reduce background, probes are often fragmented post-transcription (~200–500 nt) to improve tissue penetration (in ISH) and accessibility to structured targets. Tools such as RNAstructure, NUPACK, or OligoArray assist in probe design to minimize self-complementarity and ensure accessible binding regions.

In RNA fluorescence in situ hybridization (RNA-FISH), long RNA probes allow visualization of gene expression at the single-cell or even single-molecule level, especially when used in multiplexed formats (e.g., MERFISH, smFISH). These probes can hybridize to multiple contiguous or non-contiguous regions of a transcript, dramatically enhancing detection of low-abundance RNAs. In targeted RNA sequencing, biotinylated long probes are used to capture specific RNA species from complex samples (e.g., FFPE tissue, plasma RNA) prior to sequencing, enriching for transcripts of interest and improving sensitivity in gene expression profiling or fusion transcript detection.

Applications of long RNA probes extend to functional genomics, non-coding RNA analysis, and clinical diagnostics. They are particularly valuable in studying long non-coding RNAs (lncRNAs), which often have structured, low-expression profiles not well-captured by shorter probes. Long probes can also be engineered to include modular aptamers or photoreactive crosslinkers for studying RNA-protein interactions or subcellular localization. While challenges include probe stability, off-target hybridization, and manufacturing complexity, advances in RNA synthesis, labeling chemistry, and computational probe design have significantly expanded the power of long RNA probes as versatile tools in RNA-centric molecular biology.

Synthetic RNA standards

Synthetic RNA standards are artificially produced RNA molecules of defined sequence, length, and concentration, designed to serve as quantitative or qualitative references in RNA-based assays. Typically ranging from 100 to several thousand nucleotides, these standards are critical for calibrating assays such as quantitative reverse transcription PCR (qRT-PCR), RNA sequencing (RNA-seq), digital droplet PCR (ddPCR), Northern blotting, and in vitro diagnostics (IVDs). They mimic native RNA molecules in structure and sequence context, ensuring assay sensitivity, reproducibility, and inter-laboratory comparability, especially when detecting low-abundance or clinically relevant RNA targets.

Synthetic RNA standards are most often produced via in vitro transcription (IVT) using bacteriophage polymerases (T7, SP6, or T3) from DNA templates, which may be cloned plasmids or synthetic gene blocks. The resulting RNA transcripts may be capped (with m7GpppN or anti-reverse cap analogs), polyadenylated, or chemically modified to more accurately mimic endogenous RNA. Standards can be full-length mRNAs, non-coding RNAs, or short reference RNAs (100–500 nt) and are rigorously purified using denaturing PAGE or HPLC to remove truncated or contaminating products. Quantification is performed using UV spectrophotometry, fluorometric assays (e.g., Qubit), or digital PCR.

In RNA quantification workflows, synthetic RNA standards act as spike-in controls either exogenous (e.g., ERCC spike-ins) or synthetic versions of endogenous targets added at known concentrations to monitor RNA extraction efficiency, reverse transcription performance, or inter-sample variability. In RNA-seq, for instance, synthetic RNAs of varying lengths and GC content are used to assess library preparation biases, normalize transcript abundance, and validate isoform quantification. In molecular diagnostics, synthetic standards are crucial for limit-of-detection (LoD) determination, clinical assay validation, and lot-to-lot consistency testing.

Applications of synthetic RNA standards continue to expand across biotechnology and clinical medicine, particularly in infectious disease diagnostics (e.g., SARS-CoV-2 RT-qPCR calibration), gene therapy vector release testing, and biomanufacturing quality control. As molecular assays become more multiplexed and single-cell–oriented, synthetic RNA panels are being developed to mimic complex transcriptomes or serve as modular templates for standardizing synthetic biology workflows. Key challenges include ensuring long-term stability, preventing RNase contamination, and designing standards that faithfully reflect the complexity of endogenous RNAs. Nonetheless, synthetic RNA standards remain indispensable tools for precision and reproducibility in RNA-centric research and diagnostic applications.

Functional RNA domain libraries

Functional RNA domain libraries are collections of synthetic RNA molecules or fragments typically ranging from 100 to 500 nucleotides that encompass diverse sequence variants encoding known or putative RNA structural or functional domains. These domains may include aptamers, ribozymes, riboswitches, miRNA response elements, internal ribosome entry sites (IRES), RNA-binding protein motifs, or translational regulators. Libraries are either entirely randomized (de novo discovery) or semi-rationally designed based on conserved structural motifs or mutagenized natural elements. The primary aim is to identify functional RNA sequences capable of regulating gene expression, mediating catalysis, or interacting specifically with target molecules or proteins.

These libraries are typically synthesized using high-throughput oligonucleotide pool synthesis and then cloned into expression vectors, often within 5' or 3' untranslated regions (UTRs), introns, or independent transcription units. In some cases, in vitro transcribed RNA pools are directly screened in biochemical assays. Selection methods vary depending on the function being tested: SELEX for binding motifs, in vitro ribozyme cleavage assays for catalysis, and reporter gene screens (e.g., GFP, luciferase) for regulatory activity. Libraries can also be linked to barcodes or self-reporting sequences for multiplexed functional profiling by high-throughput sequencing.

A common application involves using functional domain libraries to discover or evolve new RNA-based regulatory elements. For example, synthetic riboswitch libraries are screened to identify ligand-binding variants that control translation in response to small molecules, enabling the creation of biosensors and logic-gated expression systems. Libraries of miRNA target sites can map miRNA-mRNA interactions or optimize synthetic gene regulation. Similarly, libraries of IRES elements can identify RNA sequences that initiate cap-independent translation, useful in stress biology and gene therapy constructs. Ribozyme libraries allow identification of novel self-cleaving elements or RNA-processing tools for synthetic biology.

The potential of functional RNA domain libraries spans multiple areas: engineering programmable RNA devices, enhancing therapeutic mRNA design, discovering new non-coding RNA elements, and building synthetic gene circuits. Challenges include ensuring structural folding fidelity, avoiding sequence bias during synthesis or amplification, and developing selection systems that recapitulate physiological conditions. With advances in machine learning–guided library design, single-cell RNA readouts, and massively parallel reporter assays (MPRAs), functional RNA domain libraries are becoming cornerstone tools in the development of next-generation RNA-based therapeutics and smart biological systems.

Linear RNA precursors for circularization

Linear RNA precursors for circularization are engineered or endogenous RNA transcripts that serve as substrates for the production of circular RNAs (circRNAs). These precursor molecules typically range from 300 to over 1,000 nucleotides, encompassing exonic and/or intronic sequences flanked by cis-acting elements that facilitate back-splicing or in vitro ligation. The essential feature of these precursors is the presence of flanking intronic repeats or structural motifs such as inverted Alu elements, complementary binding regions, or ribozyme sequences that bring the 5′ and 3′ ends of the precursor into spatial proximity, promoting the ligation of a downstream 5′ splice donor to an upstream 3′ splice acceptor to form a covalently closed RNA loop.

In natural back-splicing, circularization is mediated by the spliceosome, and the precursor must include properly oriented splice sites and accessory sequences. In synthetic systems, linear precursors are often transcribed from plasmids or PCR templates and processed either in vivo via endogenous splicing machinery or in vitro using self-splicing ribozymes (e.g., twister, hammerhead) or enzymatic ligation (e.g., T4 RNA ligase). Linear precursors can also include engineered features such as aptamer domains, internal ribosome entry sites (IRES), or protein-binding motifs that are retained post-circularization to enable functionality in translation, regulation, or molecular targeting.

Designing effective linear precursors involves optimizing several parameters: (1) ensuring efficient formation of RNA secondary structures that promote circularization; (2) minimizing cryptic splice sites or premature transcriptional termination; and (3) controlling the length and sequence of flanking introns to avoid recombination or misfolding. For in vitro applications, synthetic linear RNAs can be purified and circularized enzymatically, often followed by RNase R treatment to remove residual linear forms. Analytical validation via RT-PCR across the back-splice junction, northern blotting, or nanopore sequencing is critical to confirm successful circularization and integrity of the circRNA product.

Linear RNA precursors for circularization are central to emerging applications in RNA therapeutics, circRNA vaccines, synthetic gene circuits, and stable RNA expression systems. Compared to linear RNAs, circRNAs offer prolonged expression, resistance to exonucleases, and potential for translation when engineered with IRES or m6A elements. Therapeutically, linear precursors allow scalable production of designer circRNAs encoding therapeutic peptides, decoys, or regulatory elements. As circular RNA biology continues to expand, precise control over linear precursor design and processing will be pivotal to unlocking the full potential of circRNA-based technologies.

In Summary

Taken together, the breadth of applications enabled by synthetic RNA molecules in the 100–500 nucleotide range underscores their transformative impact on modern biology and medicine. These RNAs occupy a sweet spot: long enough to encode functional complexity binding motifs, regulatory elements, catalytic activity yet compact enough for efficient synthesis, modification, and delivery. Across domains as diverse as gene editing, immunotherapy, synthetic biology, and molecular diagnostics, this size class has enabled tools that are not only experimentally robust but also clinically actionable.

What’s striking is the modularity and versatility that these RNAs offer. A single construct can simultaneously carry targeting information, therapeutic function, and regulatory control. We’re seeing CRISPR guides with engineered scaffolds, mRNA fragments that train the immune system, and hybrid molecules that combine the best of RNA and DNA chemistry. Even the way we quantify, track, and troubleshoot biology through reporter RNAs, barcodes, and synthetic standards is increasingly reliant on this versatile format. They’ve become not just experimental tools, but the foundation of programmable molecular systems.

As the field advances, we’re moving beyond simply using RNA as a passive intermediary in gene expression. Instead, we’re beginning to think of RNA as a programmable interface a medium through which we can sense, compute, and respond to biological signals with extraordinary precision. The continued refinement of RNA structure prediction, in vivo stability, and delivery platforms will only accelerate this shift, making synthetic RNAs ever more practical and potent.

Ultimately, these mid-length RNAs have quietly become one of the most powerful molecular tools in the modern life sciences toolkit. And as we gain deeper control over how they’re designed and deployed, their role will likely expand shaping the next generation of treatments, diagnostics, and biological systems not just in theory, but in practice.