Newly emerging RNA types are gaining attention in research and therapeutic applications. Custom synthesis is expected to play a crucial role in their production. Here is a list of key RNA molecules in the expanding biotherapeutic space. These are circular RNA (circRNA), self-amplifying RNA (saRNA), long non-coding RNA (lncRNA), small activating RNA (saRNA), guide RNA (gRNA) for CRISPR Systems, RNA aptamers, mRNA with custom modifications, to name a few.
The universe of synthetic custom RNAs is still expanding. Unlike DNA, RNA can fold into various secondary and tertiary structures. Examples are stems, loops, and hairpins, which can act as regulatory structures. Since the 1970s, the RNA field has seen many advances in the study of RNA. Recent discoveries of new RNA types have vastly expanded the known "RNA world." Continuing RNA research transforms our understanding of how cells regulate genes, adapt to environments, and maintain genetic stability. Today, ongoing research continuously reveals more context-specific and regulatory RNA molecules, revealing that RNAs are not merely messengers but key regulators and actors in cellular life.
In the mid-1970s, schoolbooks knew of three types of RNA – messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). In the 1980s and early 1990s, early RNA isolation protocols did not favor efficient recovery of small transcripts; therefore, 1993 RNA was still viewed as an "interesting" molecule studied using Northern blotting.
Breakthrough technologies and paradigm-shifting insights mark the discovery of different RNA types spanning over a century. Here is a short outline highlighting milestones:
Early 20th Century to 1960s: Early Discoveries and Foundational RNAs.
Early 20th Century: In 1868, Friedrich Miescher discovered nucleic acids, but it wasn’t until later that RNA was distinguished from DNA. By the early 20th century, RNA was identified with roles suspected to involve information processing.
1950s: Messenger RNA (mRNA): In the 1950s, several researchers, including François Jacob, Jacques Monod, and Sydney Brenner, identified mRNA as the intermediary molecule carrying genetic information from DNA to ribosomes for protein synthesis.
Transfer RNA (tRNA) and Ribosomal RNA (rRNA): tRNA and rRNA were discovered and characterized as molecules involved in translation. tRNA transfers amino acids to the ribosome, while rRNA forms the structural and catalytic components of the ribosome.
1960s: Small Nuclear RNAs (snRNAs): When scientists studied the spliceosome complex, they discovered snRNAs. We now know that snRNAs play essential roles in the splicing of mRNA by removing introns from pre-mRNA sequences.
1990 to Early 2000s: Discovery of RNA Interference and Regulatory RNAs.
1990s: Micro RNA (miRNA): Discovered in C. elegans by Victor Ambros and colleagues in 1993, miRNAs were the first type of small regulatory RNA found to regulate gene expression post-transcriptionally by binding to target mRNAs.
Early 2000s: Small Interfering RNA (siRNA): In 1998, Andrew Fire and Craig Mello discovered RNA interference (RNAi) in C. elegans, where siRNAs silenced specific genes. This led to a wave of research on gene regulation, earning them the Nobel Prize in 2006.
Piwi-interacting RNA (piRNA): Identified around 2001, piRNAs are a class of small RNAs that protect the germline from transposable elements. They interact with PIWI proteins, a subfamily of the Argonaute proteins involved in gene silencing.
2000 to 2010: The RNA World expanded: Discovery of Long Non-Coding RNAs and Circular RNAs
2000s to 2010s: Long Non-Coding RNA (lncRNA): Initially observed as transcriptional "noise," lncRNAs in the early 2000s were identified as functional regulatory RNAs. However, unlike other RNAs, they do not code for proteins but regulate gene expression at the transcriptional and post-transcriptional levels.
Circular RNA (circRNA): First observed in the 1970s but rediscovered in the 2010s, circRNAs are stable circular RNA molecules formed by back-splicing which can act as miRNA sponges, regulating gene expression.
2010 to Present: Newly Discovered and Emerging RNA Types.
2010s: Enhancer RNAs (eRNAs): Identified in 2010, these RNAs are transcribed from enhancer regions in DNA. They regulate nearby genes, adding complexity to gene expression control.
Small Conditional RNA (scRNA): scRNAs are a recently identified RNA type that plays roles in specialized cellular conditions, like cellular stress responses.
Y RNA: This RNA class is now recognized for its role in DNA replication and potentially RNA quality control.
Small Nucleolar RNAs (snoRNAs) were initially identified as rRNA modifiers, but recent studies suggest they may also regulate other types of RNA and cellular processes.
2020s and Beyond: Cutting-Edge Discoveries and Functional Roles
RNA Based Thermo Sensors: These RNA molecules change conformation based on temperature, directly linking environmental conditions to gene regulation.
Antisense RNAs (ASOs): Antisense RNAs, transcribed in the opposite direction to sense mRNAs, are now recognized as gene regulators that can form double-stranded RNA, influencing mRNA stability and translation.
Context-dependent RNA
Many RNA species are context-dependent RNA types, referring to RNA molecules whose functions and characteristics change depending on the cellular or environmental context. Unlike many well-known RNAs, such as mRNA, rRNA, or tRNA, context-dependent RNAs can vary in function or interact differently with the cellular machinery based on specific signals, stressors, or developmental stages. Complex regulatory mechanisms in cells depend on RNA molecules that adjust their roles according to context. These mechanisms allow cells to respond dynamically to changing environments or internal states.
Examples of context-dependent RNA types are:
Long Non-Coding RNAs (lncRNAs): Most of lncRNAs are only expressed under certain conditions, for example, during cellular stress, tissue injury, or disease. Their roles in gene regulation, chromatin remodeling, and interaction with transcription factors can change depending on the cell type or developmental stage.
Small Nuclear RNAs (snRNAs) and Small Nucleolar RNAs (snoRNAs): These RNAs are traditionally known for their roles in RNA splicing (snRNAs) and rRNA modification (snoRNAs). Some snRNAs and snoRNAs also exhibit context-dependent functions, like interacting with specific mRNAs under stress or aiding in translational control in response to cellular changes.
miRNAs and siRNAs: These small RNAs regulate gene expression through RNA interference (RNAi) but may have different targets in different cell types, developmental stages, or environmental conditions. For example, certain miRNAs are selectively expressed or activated in response to specific signals, such as hypoxia or nutrient changes.
Circular RNAs (circRNAs): Typically generated by back-splicing, circRNAs can act as miRNA sponges, influence protein translation, or act as scaffolds. They often have tissue-specific or developmental stage-specific expression and might play roles in disease-specific conditions.
RNA Thermo Sensors: Some RNA structures act as thermo sensors that change conformation based on temperature. These conformational changes can regulate gene expression, making these RNAs context-dependent based on environmental temperature.
Stress-Induced RNAs: Some RNAs, such as certain lncRNAs and circRNAs, are upregulated under stress conditions like oxidative stress, hypoxia, or nutrient deprivation, and play roles in cellular adaptation.
Antisense RNAs: These RNAs are complementary to mRNA sequences and can regulate gene expression by forming double-stranded RNA with their target. Their activity and function can vary depending on cellular signals.
Contemporary Synthetic RNA
Many synthetic oligonucleotides are short, single- or double-stranded DNA or RNA molecules artificially synthesized in the laboratory or production facility. Oligonucleotides ranging from 15 to 100 nucleotides are generally designed for various research, diagnostic, and therapeutic applications. Unlike naturally occurring DNA or RNA, produced by living organisms, synthetic oligonucleotides are created by chemically linking individual nucleotides in a specific sequence and can contain longer sequences, also known as longmers.
Uses of Synthetic Oligonucleotides
Genetic Research: Synthetic DNA oligonucleotides are used as primers in PCR (Polymerase Chain Reaction) to amplify specific DNA regions, making it easier to study genes.
Gene Editing: In technologies like CRISPR, oligonucleotides can guide the Cas9 enzyme to precise DNA locations, allowing for targeted gene editing.
Diagnostics: Synthetic oligonucleotide probes can detect specific DNA or RNA sequences associated with pathogens or genetic mutations.
Therapeutics: Synthetic oligonucleotides allow the design of drugs targeting specific genes or cellular processes, for example, modified DNA or RNA based antisense oligonucleotides (ASOs) that bind to and block RNA transcripts to treat diseases.
Modifications
Modern automated and manual chemistries allows the synthesis and production of custom synthetic oligonucleotides, both DNA and RNA, unmodified or modified. A variety of modifications including cholesterol, fluorescent dyes, or phosphate gros, and others when incorporated into oligonucleotides increase stability, enhance cellular uptake, or improve binding properties.
Types of Custom Synthetic Oligonucleotides
DNA and RNA Oligonucleotides: Used for PCR primers, gene synthesis, antisense therapies, and RNA interference studies.
Peptide Nucleic Acids (PNAs): Synthetic analogs used in diagnostics and as probes because of their high stability and binding affinity.
Bridged Nucleic Acids (BNAs): Modified oligos used to enhance binding affinity and stability, commonly in gene expression studies and diagnostic assays.
Locked Nucleic Acids (LNAs): Modified oligos used to enhance binding affinity and stability, commonly in gene expression studies and diagnostic assays.
Morpholinos (PMOs and PPMOs): Nucleic acid analogs that block translation, often used in gene knockdown experiments.
Antisense Oligonucleotides (ASOs): Designed to modulate gene expression, used therapeutically to treat genetic diseases.
Aptamers: Short DNA or RNA oligos that can bind specific molecules, used in diagnostic assays and as drug delivery agents.
Synthetic custom oligonucleotides are used across a vast range of research and therapeutic fields. Each type is designed to meet specific molecular and functional needs. RNA molecules come in a variety of types, each with a unique structure and function. Below is an overview of the main RNA types found in cells and specialized or synthetic types:
Major Types of RNA
Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes for protein synthesis.
Transfer RNA (tRNA): Transports amino acids to the ribosome during protein synthesis.
Ribosomal RNA (rRNA): Forms the core of ribosomes and catalyzes protein synthesis.
Regulatory and Functional RNAs
MicroRNA (miRNA): MiRNAs are small RNAs that regulate gene expression by binding to mRNA, often repressing translation.
Small Interfering RNA (siRNA): SiRNA are the major players in RNA interference, a process that silences gene expression, mainly used for research and therapeutic purposes.
Long Non-Coding RNA (lncRNA): LncRNA are longer RNA molecules regulating gene expression and chromatin structure.
Small Nuclear RNA (snRNA): These RNA molecules are part of the spliceosome and essential for RNA splicing in eukaryotes.
Small Nucleolar RNA (snoRNA): Involved in modifying other RNAs, particularly rRNA, by adding methyl groups or pseudouridines.
Other Specialized RNAs
Piwi-Interacting RNA (piRNA): Protects germ cells from transposon activity by silencing transposons.
Guide RNA (gRNA): Guides RNA-editing enzymes to specific RNA sequences, often used in CRISPR/Cas9 technology.
Circular RNA (circRNA): Formed by back-splicing, they can act as molecular sponges for miRNAs and may play roles in gene regulation.
Synthetic RNAs
Antisense RNA (asRNA): Complementary to mRNA, it can block translation and is used in research and therapies.
Aptamer RNA: Synthetic RNA that can bind specific molecules, used in diagnostics and therapeutics.
Self-replicating (srRNA) or self-amplifying RNA (saRNA): Tools used in vaccines and therapeutic RNAs that can amplify small amounts of mRNA into clinical levels, derived from positive-strand DNA viruses. In these RNAs, the genes of structural proteins is replaced with genes of interest.
Viral RNAs
Viral RNA Genomes: RNA viruses have genomes made of RNA, which can be either positive-sense or negative-sense, single or double-stranded, and some include functional RNAs that manipulate host cells.
There are at least a dozen major types of RNA, but ongoing research may uncover even more specialized and context-dependent RNA types, each type playing a critical role in various biological processes and gene regulation pathways.
Reference
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Huang G, Zhang F, Xie D, Ma Y, Wang P, Cao G, Chen L, Lin S, Zhao Z, Cai Z. High-throughput profiling of RNA modifications by ultra-performance liquid chromatography coupled to complementary mass spectrometry: Methods, quality control, and applications. Talanta. 2023 Oct 1;263:124697. [Pubmed]
Huang Y, Wang J, Zhao Y, Wang H, Liu T, Li Y, Cui T, Li W, Feng Y, Luo J, Gong J, Ning L, Zhang Y, Wang D, Zhang Y. cncRNAdb: a manually curated resource of experimentally supported RNAs with both protein-coding and noncoding function. Nucleic Acids Res. 2021 Jan 8;49(D1):D65-D70. [PMC]
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National Academies of Sciences, Engineering, and Medicine; Health and Medicine Division; Division on Earth and Life Studies; Board on Health Sciences Policy; Board on Life Sciences; Toward Sequencing and Mapping of RNA Modifications Committee. Charting a Future for Sequencing RNA and Its Modifications: A New Era for Biology and Medicine. Washington (DC): National Academies Press (US); 2024 Jul 22. PMID: 39159274. [Pubmed, charting-a-future-for-sequencing-rna-and-its-modifications]
Wang, D., Fahana, A., Biochemistry, RNA Structure [books]
Yan TM, Pan Y, Yu ML, Hu K, Cao KY, Jiang ZH. Full-Range Profiling of tRNA Modifications Using LC-MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy. Anal Chem. 2021 Jan 26;93(3):1423-1432. [ Pubmed]
Yang, S., Kim, SH., Yang, E. et al. Molecular insights into regulatory RNAs in the cellular machinery. Exp Mol Med 56, 1235–1249 (2024). [Nature]
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