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A hairpin loop from a pre-mRNA. Highlighted are the nucleobases (green) and the ribose-phosphate backbone (blue). This is a single strand of RNA that folds back upon itself.
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Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyribonucleic acid (DNA) are nucleic acids. The nucleic acids constitute one of the four major macromolecules essential for all known forms of life. RNA is assembled as a chain of nucleotides. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters G, U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function in which RNA molecules direct the synthesis of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) then links amino acids together to form coded proteins.

It has become widely accepted in science[1] that early in the history of life on Earth, prior to the evolution of DNA and possibly of protein-based enzymes as well, an "RNA world" existed in which RNA served as both living organisms' storage method for genetic information—a role fulfilled today by DNA, except in the case of RNA viruses—and potentially performed catalytic functions in cells—a function performed today by protein enzymes, with the notable and important exception of the ribosome, which is a ribozyme.

Chemical structure of RNA

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Main article: Nucleic acid structure

Basic chemical composition

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Watson-Crick base pairs in a siRNA. Hydrogen atoms are not shown.

Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). Adenine and guanine are purines, and cytosine and uracil are pyrimidines. A phosphate group is attached to the 3' position of one ribose and the 5' position of the next. The phosphate groups have a negative charge each, making RNA a charged molecule (polyanion). The bases form standard hydrogen bonds between cytosine and guanine and between adenine and uracil, while guanine and uracil can pair through a non-canonical G–U wobble base pair.[2] However, other interactions are possible, such as a group of adenine bases binding to each other in a bulge,[3] or the GNRA tetraloop that has a guanine–adenine base-pair.[2]

Differences between DNA and RNA

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Three-dimensional representation of the 50S ribosomal subunit. Ribosomal RNA is in brown, proteins in blue. The active site is a small segment of rRNA, indicated in red.

The chemical structure of RNA is very similar to that of DNA, but differs in three primary ways:

  • Unlike double-stranded DNA, RNA is usually a single-stranded molecule (ssRNA)[4] in many of its biological roles and consists of much shorter chains of nucleotides.[5] However, double-stranded RNA (dsRNA) can form and (moreover) a single RNA molecule can, by complementary base pairing, form intrastrand double helixes, as in tRNA.
  • While the sugar-phosphate "backbone" of DNA contains deoxyribose, RNA contains ribose instead.[6] Ribose has a hydroxyl group attached to the pentose ring in the 2' position, whereas deoxyribose does not. The hydroxyl groups in the ribose backbone make RNA more chemically labile than DNA by lowering the activation energy of hydrolysis.
  • The complementary base to adenine in DNA is thymine, whereas in RNA, it is uracil, which is an unmethylated form of thymine.[7]

Like DNA, most biologically active RNAs, including mRNA, tRNA, rRNA, snRNAs, and other non-coding RNAs, contain self-complementary sequences that allow parts of the RNA to fold[8] and pair with itself to form double helices. Analysis of these RNAs has revealed that they are highly structured. Unlike DNA, their structures do not consist of long double helices, but rather collections of short helices packed together into structures akin to proteins.

In this fashion, RNAs can achieve chemical catalysis (like enzymes).[9] For instance, determination of the structure of the ribosome—an RNA-protein complex that catalyzes the assembly of proteins—revealed that its active site is composed entirely of RNA.[10]

Structure of a fragment of an RNA, showing a guanosyl subunit

An important structural component of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar. The presence of this functional group causes the helix to mostly take the A-form geometry,[11] although in single strand dinucleotide contexts, RNA can rarely also adopt the B-form most commonly observed in DNA.[12] The A-form geometry results in a very deep and narrow major groove and a shallow and wide minor groove.[13] A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave the backbone.[14]

Secondary and tertiary structures

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The functional form of single-stranded RNA molecules, just like proteins, frequently requires a specific spatial tertiary structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops, bulges, and internal loops.[15] In order to create, i.e., design, RNA for any given secondary structure, two or three bases would not be enough, but four bases are enough.[16] This is likely why nature has "chosen" a four base alphabet: fewer than four would not allow the creation of all structures, while more than four bases are not necessary to do so. Since RNA is charged, metal ions such as Mg2+ are needed to stabilise many secondary and tertiary structures.[17]

The naturally occurring enantiomer of RNA is D-RNA composed of D-ribonucleotides. All chirality centers are located in the D-ribose. By the use of L-ribose or rather L-ribonucleotides, L-RNA can be synthesized. L-RNA is much more stable against degradation by RNase.[18]

Like other structured biopolymers such as proteins, one can define topology of a folded RNA molecule. This is often done based on arrangement of intra-chain contacts within a folded RNA, termed as circuit topology.

Chemical modifications

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Secondary structure of a telomerase RNA

RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil),[19] but these bases and attached sugars can be modified in numerous ways as the RNAs mature. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T) are found in various places (the most notable ones being in the TΨC loop of tRNA).[20] Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine (I). Inosine plays a key role in the wobble hypothesis of the genetic code.[21]

There are more than 100 other naturally occurring modified nucleosides.[22] The greatest structural diversity of modifications can be found in tRNA,[23] while pseudouridine and nucleosides with 2'-O-methylribose often present in rRNA are the most common.[24] The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that, in ribosomal RNA, many of the post-transcriptional modifications occur in highly functional regions, such as the peptidyl transferase center[25] and the subunit interface, implying that they are important for normal function.[26]

Types of RNA

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See also: List of RNAs
Structure of a hammerhead ribozyme, a ribozyme that cuts RNA

Messenger RNA (mRNA) is the type of RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell cytoplasm. The coding sequence of the mRNA determines the amino acid sequence in the protein that is produced.[27] However, many RNAs do not code for protein (about 97% of the transcriptional output is non-protein-coding in eukaryotes[28][29][30][31]).

These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.[32] The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.[7] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,[33] and the catalysis of peptide bond formation in the ribosome;[10] these are known as ribozymes.

According to the length of RNA chain, RNA includes small RNA and long RNA.[34] Usually, small RNAs are shorter than 200 nt in length, and long RNAs are greater than 200 nt long.[35] Long RNAs, also called large RNAs, mainly include long non-coding RNA (lncRNA) and mRNA. Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA)[36] and small rDNA-derived RNA (srRNA).[37] There are certain exceptions as in the case of the 5S rRNA of the members of the genus Halococcus (Archaea), which have an insertion, thus increasing its size.[38][39][40]

RNAs involved in protein synthesis

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Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) corresponds to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain amount of time, the message degrades into its component nucleotides with the assistance of ribonucleases.[27]

Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding.[32]

A diagram of how mRNA is used to create polypeptide chains

Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. The rRNA is the component of the ribosome that hosts translation. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time.[27] Nearly all the RNA found in a typical eukaryotic cell is rRNA.

Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.[41]

Regulatory RNA

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The earliest known regulators of gene expression were proteins known as repressors and activators – regulators with specific short binding sites within enhancer regions near the genes to be regulated.[42]  Later studies have shown that RNAs also regulate genes. There are several kinds of RNA-dependent processes in eukaryotes regulating the expression of genes at various points, such as RNA interference repressing genes post-transcriptionally, long non-coding RNAs shutting down blocks of chromatin epigenetically, and enhancer RNAs inducing increased gene expression.[43] Bacteria and archaea have also been shown to use regulatory RNA systems such as bacterial small RNAs and CRISPR.[44] Fire and Mello were awarded the 2006 Nobel Prize in Physiology or Medicine for discovering microRNAs (miRNAs), specific short RNA molecules that can base-pair with mRNAs.[45]

MicroRNA (miRNA) and small interfering RNA (siRNA)

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See also: RNA interference

Post-transcriptional expression levels of many genes can be controlled by RNA interference, in which miRNAs, specific short RNA molecules, pair with mRNA regions and target them for repression.[46] RNA interference is carried out by the RNA-induced silencing complex, which uses a miRNA guide to regulate complementary mRNAs through base pairing interactions and results in either degradaing the mRNA or blocking its translation.[47]

Long non-coding RNAs

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See also: Long Non-coding RNA

Next to be linked to regulation were Xist and other long noncoding RNAs associated with X chromosome inactivation.  Their roles, at first mysterious, were shown by Jeannie T. Lee and others to be the silencing of blocks of chromatin via recruitment of Polycomb complex so that messenger RNA could not be transcribed from them.[48] Additional lncRNAs, currently defined as RNAs of more than 200 base pairs that do not appear to have coding potential,[49] have been found associated with regulation of stem cell pluripotency and cell division.[49]

Enhancer RNAs

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See also: Enhancer RNA

The third major group of regulatory RNAs is called enhancer RNAs.[49]  It is not clear at present whether they are a unique category of RNAs of various lengths or constitute a distinct subset of lncRNAs.  In any case, they are transcribed from enhancers, which are known regulatory sites in the DNA near genes they regulate.[49][50]  They up-regulate the transcription of the gene(s) under control of the enhancer from which they are transcribed.[49][51]

Small RNA in prokaryotes

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Small RNA

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At first, regulatory RNA was thought to be a eukaryotic phenomenon, a part of the explanation for why so much more transcription in higher organisms was seen than had been predicted. But as soon as researchers began to look for possible RNA regulators in bacteria, they turned up there as well, termed as small RNA (sRNA).[52][44] Currently, the ubiquitous nature of systems of RNA regulation of genes has been discussed as support for the RNA World theory.[43][53] There are indications that the enterobacterial sRNAs are involved in various cellular processes and seem to have significant role in stress responses such as membrane stress, starvation stress, phosphosugar stress and DNA damage. Also, it has been suggested that sRNAs have been evolved to have important role in stress responses because of their kinetic properties that allow for rapid response and stabilisation of the physiological state.[4] Bacterial small RNAs generally act via antisense pairing with mRNA to down-regulate its translation, either by affecting stability or affecting cis-binding ability.[43] Riboswitches have also been discovered. They are cis-acting regulatory RNA sequences acting allosterically. They change shape when they bind metabolites so that they gain or lose the ability to bind chromatin to regulate expression of genes.[54][55]

CRISPR RNA

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Archaea also have systems of regulatory RNA.[56] The CRISPR system, recently being used to edit DNA in situ, acts via regulatory RNAs in archaea and bacteria to provide protection against virus invaders.[43][57]

RNA synthesis and processing

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Synthesis

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Synthesis of RNA typically occurs in the cell nucleus and is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3' to 5' direction, synthesizing a complementary RNA molecule with elongation occurring in the 5' to 3' direction. The DNA sequence also dictates where termination of RNA synthesis will occur.[58]

Primary transcript RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA and introns are removed by the spliceosome.

There are also a number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material.[59] Also, RNA-dependent RNA polymerase is part of the RNA interference pathway in many organisms.[60]

RNA processing

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Uridine to pseudouridine is a common RNA modification.

Many RNAs are involved in modifying other RNAs. Introns are spliced out of pre-mRNA by spliceosomes, which contain several small nuclear RNAs (snRNA),[7] or the introns can be ribozymes that are spliced by themselves.[61] RNA can also be altered by having its nucleotides modified to nucleotides other than A, C, G and U. In eukaryotes, modifications of RNA nucleotides are in general directed by small nucleolar RNAs (snoRNA; 60–300 nt),[32] found in the nucleolus and cajal bodies. snoRNAs associate with enzymes and guide them to a spot on an RNA by basepairing to that RNA. These enzymes then perform the nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the target of base modification.[62][63] RNA can also be methylated.[64][65]


RNA in genetics

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RNA genomes

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Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the genome as the virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated by a host plant cell's polymerase.[66]

Reverse transcription

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Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one another,[67] and telomerase contains an RNA that is used as template for building the ends of eukaryotic chromosomes.[68]

Double-stranded RNA

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Double-stranded RNA
Main article: Double-stranded RNA

Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells, but with the replacement of thymine by uracil and the adding of one oxygen atom. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA, such as viral RNA or siRNA, can trigger RNA interference in eukaryotes, as well as interferon response in vertebrates.[69][70][71][72] In eukaryotes, double-stranded RNA (dsRNA) plays a role in the activation of the innate immune system against viral infections.[73]

Circular RNA

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Main article: Circular RNA

In the late 1970s, it was shown that there is a single stranded covalently closed, i.e. circular form of RNA expressed throughout the animal and plant kingdom (see circRNA).[74] circRNAs are thought to arise via a "back-splice" reaction where the spliceosome joins a upstream 3' acceptor to a downstream 5' donor splice site. So far the function of circRNAs is largely unknown, although for few examples a microRNA sponging activity has been demonstrated.

Key discoveries in RNA biology

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Further information: History of RNA biology
Robert W. Holley, left, poses with his research team.

Research on RNA has led to many important biological discoveries and numerous Nobel Prizes. Nucleic acids were discovered in 1868 by Friedrich Miescher, who called the material 'nuclein' since it was found in the nucleus.[75] It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis was suspected already in 1939.[76] Severo Ochoa won the 1959 Nobel Prize in Medicine (shared with Arthur Kornberg) after he discovered an enzyme that can synthesize RNA in the laboratory.[77] However, the enzyme discovered by Ochoa (polynucleotide phosphorylase) was later shown to be responsible for RNA degradation, not RNA synthesis. In 1956 Alex Rich and David Davies hybridized two separate strands of RNA to form the first crystal of RNA whose structure could be determined by X-ray crystallography.[78]

The sequence of the 77 nucleotides of a yeast tRNA was found by Robert W. Holley in 1965,[79] winning Holley the 1968 Nobel Prize in Medicine (shared with Har Gobind Khorana and Marshall Nirenberg).

In the early 1970s, retroviruses and reverse transcriptase were discovered, showing for the first time that enzymes could copy RNA into DNA (the opposite of the usual route for transmission of genetic information). For this work, David Baltimore, Renato Dulbecco and Howard Temin were awarded a Nobel Prize in 1975. In 1976, Walter Fiers and his team determined the first complete nucleotide sequence of an RNA virus genome, that of bacteriophage MS2.[80]

In 1977, introns and RNA splicing were discovered in both mammalian viruses and in cellular genes, resulting in a 1993 Nobel to Philip Sharp and Richard Roberts. Catalytic RNA molecules (ribozymes) were discovered in the early 1980s, leading to a 1989 Nobel award to Thomas Cech and Sidney Altman. In 1990, it was found in Petunia that introduced genes can silence similar genes of the plant's own, now known to be a result of RNA interference.[81][82]

At about the same time, 22 nt long RNAs, now called microRNAs, were found to have a role in the development of C. elegans.[83] Studies on RNA interference earned a Nobel Prize for Andrew Fire and Craig Mello in 2006, and another Nobel for studies on the transcription of RNA to Roger Kornberg in the same year. The discovery of gene regulatory RNAs has led to attempts to develop drugs made of RNA, such as siRNA, to silence genes.[84] Adding to the Nobel prizes for research on RNA, in 2009 it was awarded for the elucidation of the atomic structure of the ribosome to Venki Ramakrishnan, Thomas A. Steitz, and Ada Yonath. In 2023 the Nobel Prize in Physiology or Medicine was awarded to Katalin Karikó and Drew Weissman for their discoveries concerning modified nucleosides that enabled the development of effective mRNA vaccines against COVID-19.[85][86][87]

Relevance for prebiotic chemistry and abiogenesis

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In 1968, Carl Woese hypothesized that RNA might be catalytic and suggested that the earliest forms of life (self-replicating molecules) could have relied on RNA both to carry genetic information and to catalyze biochemical reactions—an RNA world.[88][89] In May 2022, scientists discovered that RNA can form spontaneously on prebiotic basalt lava glass, presumed to have been abundant on the early Earth.[90][91]

In March 2015, DNA and RNA nucleobases, including uracil, cytosine and thymine, were reportedly formed in the laboratory under outer space conditions, using starter chemicals such as pyrimidine, an organic compound commonly found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), is one of the most carbon-rich compounds found in the universe and may have been formed in red giants or in interstellar dust and gas clouds.[92] In July 2022, astronomers reported massive amounts of prebiotic molecules, including possible RNA precursors, in the galactic center of the Milky Way Galaxy.[93][94]

Medical applications

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RNA, initially deemed unsuitable for therapeutics due to its short half-life, has been made useful through advances in stabilization. Therapeutic applications arise as RNA folds into complex conformations and binds proteins, nucleic acids, and small molecules to form catalytic centers.[95] RNA-based vaccines are thought to be easier to produce than traditional vaccines derived from killed or altered pathogens, because it can take months or years to grow and study a pathogen and determine which molecular parts to extract, inactivate, and use in a vaccine. Small molecules with conventional therapeutic properties can target RNA and DNA structures, thereby treating novel diseases. However, research is scarce on small molecules targeting RNA and approved drugs for human illness. Ribavirin, branaplam, and ataluren are currently available medications that stabilize double-stranded RNA structures and control splicing in a variety of disorders.[96][97]

Protein-coding mRNAs have emerged as new therapeutic candidates, with RNA replacement being particularly beneficial for brief but torrential protein expression.[98] In vitro transcribed mRNAs (IVT-mRNA) have been used to deliver proteins for bone regeneration, pluripotency, and heart function in animal models.[99][100][101][102][103] SiRNAs, short RNA molecules, play a crucial role in innate defense against viruses and chromatin structure. They can be artificially introduced to silence specific genes, making them valuable for gene function studies, therapeutic target validation, and drug development.[98]

mRNA vaccines have emerged as an important new class of vaccines, using mRNA to manufacture proteins which provoke an immune response. Their first successful large-scale application came in the form of COVID-19 vaccines during the COVID-19 pandemic.

See also

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References

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RNA

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from Grokipedia
Ribonucleic acid (RNA) is a linear polymer of ribonucleotides, each consisting of a ribose sugar, a phosphate group, and one of four nitrogenous bases—adenine (A), cytosine (C), guanine (G), or uracil (U)—that plays central roles in coding, decoding, regulation, and expression of genes in most living organisms and many viruses. Unlike deoxyribonucleic acid (DNA), RNA is typically single-stranded, allowing it to fold into complex three-dimensional structures that enable its diverse functions, though some RNA viruses contain double-stranded forms as their genetic material. The of RNA features a backbone formed by 5' to 3' phosphodiester bonds between the sugars and phosphates, with the 2' hydroxyl group on distinguishing it from DNA's and contributing to RNA's reactivity and flexibility. Bases pair via hydrogen bonds—A with U (two bonds) and C with G (three bonds)—creating secondary structures such as stems, loops, and hairpins, while tertiary folding into helices, bulges, and pseudoknots supports specific interactions with proteins, other RNAs, and small molecules. RNA exists in multiple types, each with specialized structures and roles; the three primary types involved in protein synthesis are messenger RNA (mRNA), which is transcribed from DNA and carries the genetic code to ribosomes as a single-stranded chain averaging 1,000–10,000 nucleotides; transfer RNA (tRNA), a cloverleaf-shaped molecule of about 70–90 nucleotides that decodes mRNA codons via its anticodon loop to deliver specific amino acids; and ribosomal RNA (rRNA), which comprises the structural and catalytic core of ribosomes (70S in prokaryotes, 80S in eukaryotes) and accounts for up to 80% of cellular RNA. Beyond these, non-coding RNAs include small nuclear RNAs (snRNAs), such as U1–U6, which form spliceosomes to process pre-mRNA by removing introns; small nucleolar RNAs (snoRNAs), which guide chemical modifications like methylation and pseudouridylation on rRNA, tRNA, and snRNA in the nucleolus; and regulatory RNAs like microRNAs (miRNAs) and small interfering RNAs (siRNAs), which mediate gene silencing through RNA interference. Other notable types encompass long non-coding RNAs (lncRNAs) that influence chromatin structure and transcription, circular RNAs that act as miRNA sponges or regulators, and ribozymes—catalytic RNAs capable of self-splicing or cleaving phosphodiester bonds. The core function of RNA is to bridge DNA's genetic information to functional proteins via transcription (DNA to mRNA) and translation (mRNA to polypeptide chains on ribosomes), a process essential for cellular and growth. RNAs also regulate by modulating transcription, mRNA stability, and translation efficiency, as seen in miRNA-mediated repression or lncRNA scaffolding of protein complexes. Certain RNAs exhibit enzymatic activity as ribozymes, facilitating reactions like formation in ribosomes or removal, while in RNA viruses, RNA serves directly as the heritable replicated by RNA-dependent RNA polymerases. Dysfunctions in RNA processing or structure underlie diseases such as and certain cancers, highlighting RNA's therapeutic potential, including in mRNA vaccines that encode antigens to elicit immune responses.

Structure and Composition

Nucleotide Components

RNA is a linear composed of repeating units known as , which are linked together to form the chain. Each consists of three primary components: a sugar called , a group, and one of four nitrogenous bases— (A), (G), (C), or uracil (U). The nitrogenous bases are heterocyclic aromatic compounds; and are purines with a fused double-ring structure, while and uracil are pyrimidines featuring a single six-membered ring. These bases attach to the sugar via an N-glycosidic bond at the C1' position of the sugar and the N9 (for purines) or N1 (for pyrimidines) of the base. The ribose sugar in RNA is specifically β-D-ribofuranose, existing in a five-membered furanose ring conformation with hydroxyl groups at the 2', 3', and 5' positions. This configuration arises from the furanose form of D-ribose, where the ring oxygen is between C1' and C4', and the β-anomer has the base attached above the plane of the ring. The distinctive 2'-hydroxyl (2'-OH) group on the ribose enhances RNA's chemical reactivity compared to deoxyribose in DNA, as it participates in nucleophilic attacks that facilitate processes like self-cleavage or enzymatic modifications. For instance, the chemical formula of adenosine monophosphate (AMP), the ribonucleotide containing adenine, is C10_{10}H14_{14}N5_5O7_7P, illustrating the integration of these components. Similar formulas apply to the other ribonucleotides: guanosine monophosphate (GMP, C10_{10}H14_{14}N5_5O8_8P), cytidine monophosphate (CMP, C9_9H14_{14}N3_3O8_8P), and uridine monophosphate (UMP, C9_9H13_{13}N2_2O9_9P). The phosphodiester backbone of RNA forms through covalent bonds between the phosphate group of one ribonucleotide and the sugar of the adjacent one, creating a directional chain. Specifically, the 3'-OH of the ribose in one nucleotide reacts with the 5'-phosphate attached to the adjacent nucleotide, forming a phosphodiester linkage and resulting in a polymer with 5' to 3' polarity. This asymmetry imparts directionality to the RNA strand, with the 5' end typically bearing a phosphate or triphosphate group and the 3' end terminating in a hydroxyl. The 2'-OH group contributes to RNA's relative instability, rendering it susceptible to hydrolysis under basic conditions; the hydroxyl acts as a nucleophile to attack the adjacent phosphodiester bond, forming a 2',3'-cyclic phosphate intermediate that leads to chain cleavage. This reactivity contrasts with the stability of DNA and underscores RNA's transient role in cellular processes.

Differences from DNA

One key chemical difference between RNA and DNA lies in the substitution of uracil (U) for (T) as one of the nitrogenous bases in RNA. Uracil pairs with (A) through two bonds, similar to the A-T pairing in DNA, whereas (G) pairs with (C) via three bonds in both molecules, contributing to the overall stability of base pairing. DNA uses instead of uracil to allow detection and repair of cytosine , which produces uracil; if DNA used uracil, such damage would be indistinguishable from normal bases. In RNA, uracil is used as it is energetically cheaper to synthesize (lacking the ) and RNA's short lifespan reduces the impact of mutations. Another fundamental distinction is the sugar component: RNA incorporates , which has a hydroxyl group (-OH) at the 2' position of the sugar ring, whereas DNA uses lacking this group. The 2'-OH group in RNA enhances the conformational flexibility of the single-stranded molecule, allowing it to adopt diverse shapes more readily than the more rigid DNA backbone. However, this same group renders RNA more vulnerable to enzymatic degradation and chemical , as it can participate in nucleophilic attacks on the phosphodiester backbone, briefly tying back to the nucleotide's inherent reactivity. RNA is typically single-stranded, in contrast to DNA's double-helical structure, which impacts their respective stability and . The absence of a complementary strand in RNA reduces base-pairing protection, making it less stable and more prone to unfolding or degradation, while DNA's double helix provides greater and resistance to environmental damage for long-term genetic storage. This single-stranded nature also facilitates RNA's role as a transient intermediary rather than a permanent . RNA molecules are generally much shorter in length than DNA, with most ranging from hundreds to thousands of , compared to DNA's genome-spanning millions. Consequently, RNA exhibits a higher turnover rate, with half-lives often ranging from minutes to hours depending on the type and cellular conditions, enabling rapid , whereas DNA persists stably across cell generations for archival purposes. Physicochemically, RNA demonstrates higher solubility in water than DNA, attributable to the polar 2'-OH group increasing hydrophilicity. This feature, combined with the single-stranded form, promotes RNA's tendency to form complex intramolecular folds through base pairing and stacking interactions, unlike the more uniform double helix of DNA. Additionally, RNA's susceptibility to degradation by ubiquitous ribonucleases (RNases) far exceeds DNA's vulnerability to deoxyribonucleases (DNases), underscoring its ephemeral nature in cellular environments.

Folding and Higher-Order Structures

The primary structure of RNA, defined by its linear of ribonucleotides, encodes the intrinsic potential for folding into functional three-dimensional conformations through specific base interactions. This dictates the locations and types of complementary bases available for , influencing the stability and architecture of higher-order structures. Seminal studies have established that variations in primary can profoundly alter folding pathways and final structures, underscoring the as the foundational determinant of RNA's conformational landscape. RNA secondary structures emerge from intramolecular base pairing along the primary chain, forming double-stranded helices interspersed with single-stranded regions. Canonical Watson-Crick base pairs (A-U and G-C) provide the core stability via two or three hydrogen bonds, respectively, while non-canonical interactions, such as the G-U wobble pair, introduce flexibility and are nearly as thermodynamically stable as Watson-Crick pairs due to their isomorphic geometry and comparable hydrogen bonding. These pairings create characteristic motifs, including stem-loops (where a helical stem terminates in a loop), hairpins (short stems closed by loops of 3-7 nucleotides), bulges (unpaired bases on one side of a helix), and internal loops (unpaired regions on both sides). Such elements allow RNA to adopt compact, hierarchical architectures that serve as scaffolds for tertiary folding.00112-X) Tertiary structures result from the spatial organization of multiple secondary elements through long-range contacts, yielding compact globular forms essential for RNA function. Key motifs include pseudoknots, in which a single-stranded loop from one stem pairs with a distant to interlock helices; coaxial helices, where adjacent helical segments stack continuously without interruption; and kissing loops, involving reciprocal base pairing between the loops of two hairpins. These interactions enable complex topologies, as exemplified by the L-shaped tertiary fold in tRNA, which positions its acceptor and anticodon arms orthogonally, or the precisely organized active sites in ribozymes that catalyze formation. The driving forces behind RNA folding encompass a balance of enthalpic and entropic contributions from non-covalent interactions. Hydrogen bonding between base edges stabilizes paired regions, while base stacking—arising from van der Waals and hydrophobic forces between adjacent aromatic bases—provides the dominant stabilization in helical segments, contributing up to 50% of the free energy in double-stranded RNA. Electrostatic repulsion from the negatively charged backbone is mitigated by divalent cations like Mg²⁺ ions, which bind specifically to facilitate tertiary contacts and neutralize charges, often increasing folding efficiency by orders of magnitude in physiological conditions.00112-X) Predicting RNA folding computationally relies on thermodynamic models that approximate free energy minimization. Algorithms like mfold, introduced in seminal work using dynamic programming to enumerate suboptimal structures, and the ViennaRNA package, which implements partition function calculations for ensemble predictions, model secondary structures by scoring base pairs based on nearest-neighbor parameters derived from melting experiments. These tools excel for sequences up to several hundred but face limitations from kinetic traps—local energy minima that trap RNA in metastable states during folding, as opposed to the global minimum—leading to discrepancies between predicted and native structures . Advanced extensions incorporate barrier-crossing heuristics to address these kinetic effects.

Chemical Modifications

RNA molecules undergo a diverse array of post-transcriptional chemical modifications that alter their structure and function, building upon the core components of , , , , and . Over 170 distinct types of these modifications have been identified as of 2025, with the majority occurring in eukaryotic organisms and prominently in (rRNA), (tRNA), and (mRNA).30638-4) These modifications include base methylations, such as N6-methyladenosine (m6A) on , pseudouridylation where is isomerized to (Ψ), and ribose 2'-O-methylation on the sugar backbone. Among these, m6A stands out as the most abundant internal modification in eukaryotic mRNA, often found in the DRACH (where D = A/G/U, R = A/G, H = A/C/U). The installation, removal, and interpretation of these modifications are mediated by enzymatic complexes known as writers, erasers, and readers, respectively. For m6A, the primary writer is the METTL3-METTL14-WTAP methyltransferase complex, which catalyzes the addition of a to the N6 position of . Erasers, such as the demethylase FTO, reverse this modification by oxidative demethylation, thereby dynamically regulating m6A levels. Readers, including YTH-domain-containing proteins like YTHDF2, recognize and bind to modified sites to influence downstream RNA processes, such as directing m6A-marked transcripts to decay pathways. Similar machinery exists for other modifications; for instance, pseudouridine synthases (e.g., enzymes) act as writers for Ψ without requiring erasers, while fibrillarin catalyzes site-specific 2'-O-methylations in rRNA. These chemical alterations profoundly impact RNA by enhancing stability against degradation, modulating base-pairing interactions, and fine-tuning processes like splicing and efficiency. For example, m6A promotes mRNA decay through YTHDF2-mediated recruitment to , thereby reducing protein output, while 2'-O-methylation stabilizes RNA structures and improves translational fidelity in rRNA. Pseudouridylation enhances RNA flexibility and stability, facilitating proper tRNA anticodon recognition during and influencing splice site selection in pre-mRNA. Such modifications collectively regulate at multiple levels, with disruptions linked to diseases including cancer and neurological disorders. RNA modifications exhibit evolutionary conservation, particularly in essential RNAs like tRNA and rRNA, where core sites such as m6A in transcripts are preserved from to humans, underscoring their fundamental roles in cellular homeostasis.00451-2) Detection of these modifications has advanced through epitranscriptomics, employing techniques like for quantitative profiling of abundant RNAs and sequencing-based methods, such as m6A-seq or Pseudo-seq, which use pulldowns or chemical labeling to map modification sites genome-wide with single-nucleotide resolution. These approaches have revealed dynamic, context-dependent modification patterns that respond to cellular stresses and developmental cues.00147-7)

Synthesis and Processing

Transcription Mechanism

Transcription is the enzymatic process by which RNA is synthesized from template, involving the of ribonucleotides in the 5' to 3' direction to produce a complementary RNA strand. This DNA-directed synthesis uses the DNA as a template, where the reads one strand (the template strand) and assembles RNA using triphosphates (NTPs) that match the complementary bases. In prokaryotes, a single , composed of core subunits and a for promoter recognition, catalyzes the transcription of all RNA types. In eukaryotes, three distinct nuclear RNA polymerases perform specialized roles: (Pol I) transcribes most ribosomal RNAs (rRNAs), (Pol II) synthesizes messenger RNAs (mRNAs) and small nuclear RNAs (snRNAs), and (Pol III) produces transfer RNAs (tRNAs) and 5S rRNA. The transcription process occurs in three main stages: initiation, elongation, and termination. Initiation begins with the binding of RNA polymerase to promoter elements on the DNA. In prokaryotes, the core promoter includes the -35 box (TTGACA consensus) and -10 box (TATAAT consensus), recognized by the sigma factor to unwind DNA and form the open complex. In eukaryotes, Pol II initiation involves the TATA box (TATAAA consensus, located 25-35 bases upstream of the start site), bound by the TATA-binding protein (TBP) as part of the transcription factor IID (TFIID) complex, which recruits additional factors and the polymerase. Eukaryotic promoters may also include enhancers, distal regulatory sequences that boost transcription rates. During elongation, the RNA polymerase moves along the DNA template, incorporating NTPs (ATP, GTP, CTP, UTP) complementary to the template bases, extending the RNA chain in the 5' to 3' direction at rates of about 20-50 per second in prokaryotes and 22-25 per second for Pol II in eukaryotes. The energy for this comes from the of the high-energy phosphoanhydride bonds in NTPs, releasing (PPi) and driving the irreversible addition of each . Fidelity is maintained through base-pairing selectivity and mechanisms; the initial misincorporation error rate is approximately 1 in 10^4 , improved by intrinsic cleavage activity in some polymerases, such as Pol III, which removes mismatched 3' termini via hydrolytic , enhancing accuracy by up to 10^3-fold. Overall transcription error rates reach about 10^{-5} per in like E. coli. Termination signals the end of RNA synthesis and release of the transcript. In prokaryotes, involves the formation of a GC-rich loop in the RNA followed by a run of uracils, causing pausing and dissociation, while rho-dependent termination uses the Rho helicase protein to unwind the RNA-DNA hybrid. Eukaryotic termination for Pol II occurs downstream of the signal, involving cleavage and release, though mechanisms vary by type.

Post-Transcriptional Processing

Post-transcriptional encompasses a series of modifications that transform the primary RNA transcript, known as pre-mRNA in eukaryotes, into mature, functional RNA molecules. These steps occur in the nucleus and are crucial for RNA stability, export to the , and proper . In prokaryotes, processing is minimal due to coupled transcription and translation, whereas eukaryotic processing is more elaborate to accommodate larger genomes and regulatory complexity. One of the initial modifications is 5' , which involves the addition of a 7-methylguanosine to the 5' end of the nascent pre-mRNA shortly after transcription initiation. This is covalently linked via a 5'-5' triphosphate bridge by the guanylyltransferase, followed by . The protects the RNA from 5' exonucleases, facilitates nuclear export through interactions with export factors like NXF1, and enhances translation initiation by recruiting the eIF4E. At the 3' end, occurs after cleavage of the pre-mRNA at a specific site defined by the AAUAAA signal, followed by the addition of a poly-A tail consisting of 200-250 residues in eukaryotes. This process is catalyzed by a multiprotein complex including cleavage and specificity factor (CPSF) and poly-A polymerase (PAP). The poly-A tail increases mRNA stability by preventing degradation from 3' exonucleases and promotes export and translation efficiency via binding to poly-A binding proteins (PABPs). Splicing removes non-coding introns and joins coding to form mature mRNA, a process mediated by the in eukaryotes, which assembles from small nuclear ribonucleoproteins (snRNPs) U1 through U6. The recognizes conserved splice sites (GU at the 5' end and AG at the 3' end of introns) and catalyzes two reactions to excise introns. Some introns, such as group I and II, can self-splice without proteins, relying on RNA . , where different exon combinations are selected, generates multiple protein isoforms from a single , expanding proteomic diversity. RNA editing introduces base changes post-transcriptionally, with adenosine-to-inosine (A-to-I) editing being prevalent in eukaryotes, performed by enzymes that deaminate to , which is read as during . This can alter codons, potentially changing or creating stop codons, thus modulating protein function and diversity. For instance, editing in transcripts affects calcium permeability in neurons. mechanisms ensure only properly processed RNAs proceed, with (NMD) targeting transcripts containing premature termination codons for degradation. NMD involves recognition by factors like UPF1, UPF2, and UPF3 during the pioneer round of , preventing accumulation of truncated proteins. This pathway degrades about 5-30% of human transcripts, highlighting its role in regulating . Secondary structures in the RNA can influence transcription termination signals, thereby affecting the substrate for these processing events.

Major Types and Functions

Protein-Coding RNAs

Protein-coding RNAs, primarily messenger RNAs (mRNAs), serve as the intermediary molecules that convey genetic information from DNA to ribosomes for protein synthesis, embodying a core aspect of the central dogma of molecular biology. In eukaryotic cells, mRNAs are typically monocistronic, encoding a single protein from one open reading frame, whereas prokaryotic mRNAs are often polycistronic, allowing multiple proteins to be translated from a single transcript organized into operons. The structure of mRNA includes a 5' untranslated region (UTR) that regulates translation initiation, a central coding sequence composed of nucleotide triplets known as codons that specify amino acid sequences, and a 3' UTR that influences mRNA stability, localization, and translation efficiency, often ending with a poly-A tail in eukaryotes. The of mRNA into proteins occurs in three main stages: , elongation, and termination. During in eukaryotes, the small ribosomal subunit binds to the 5' cap of the mRNA with assistance from eukaryotic initiation factors (eIFs), scanning to the (AUG) recognized via the for efficient assembly of the full . Elongation follows as transfer RNAs (tRNAs) match their anticodons to successive mRNA codons in the 's A site, facilitating formation and translocation along the mRNA. Termination is triggered by stop codons (UAA, UAG, UGA) in the A site, prompting release factors to disassemble the and liberate the nascent polypeptide. Following transcription in the nucleus, mature eukaryotic mRNAs are exported to the through nuclear pore complexes, where they localize to specific cellular compartments for targeted , such as dendrites in neurons. mRNA stability is tightly regulated, with degradation initiated by deadenylation (shortening of the poly-A tail) followed by and exonucleolytic digestion, ensuring rapid turnover of transcripts in response to cellular needs. In terms of abundance, mRNAs constitute approximately 1-5% of total cellular RNA in eukaryotes, with half-lives ranging from minutes for short-lived transcripts like the proto-oncogene c-fos to several hours for more stable ones, reflecting their role in dynamic control. Evolutionarily, protein-coding RNAs are thought to trace back to an ancient , where self-replicating RNA molecules encoded rudimentary peptides via a primitive , laying the foundation for modern systems. Prior to translation, mRNA precursors undergo processing steps like capping, splicing, and to generate functional transcripts.

Regulatory Non-Coding RNAs

Regulatory non-coding RNAs (ncRNAs) are a diverse class of RNA molecules that do not encode proteins but play crucial roles in modulating at transcriptional, post-transcriptional, and epigenetic levels. These RNAs, ranging from short 20-30 species to long transcripts exceeding 200 nucleotides, interact with DNA, RNA, or proteins to fine-tune cellular processes such as development, differentiation, and response to stress. Unlike protein-coding RNAs, their primary function lies in regulation rather than translation, enabling precise control over genome activity without altering the genetic code directly. MicroRNAs (miRNAs) are small endogenous ncRNAs approximately 21-25 in length that primarily repress post-transcriptionally. They are initially transcribed as primary miRNAs (pri-miRNAs) with stem-loop structures, which are processed in the nucleus by the complex containing and DGCR8 to form precursor miRNAs (pre-miRNAs). These precursors are then exported to the cytoplasm and cleaved by into mature miRNAs, which are loaded into the (RISC) containing proteins. Within RISC, miRNAs typically bind to the 3' untranslated regions (UTRs) of target mRNAs through partial base-pairing, leading to translational repression or mRNA destabilization and decay. This mechanism allows a single miRNA to regulate hundreds of targets, influencing processes like and ; for instance, the founding miRNA lin-4 was discovered in C. elegans where it negatively regulates LIN-14 protein levels during development. Small interfering RNAs (siRNAs) are structurally similar to miRNAs, also 20-25 nucleotides long and processed by , but they arise primarily from exogenous double-stranded RNA (dsRNA) precursors and mediate sequence-specific (RNAi) for . Unlike miRNAs, siRNAs often exhibit perfect complementarity to their targets, triggering direct cleavage by Argonaute-2 in the RISC complex rather than translational repression. This pathway was first demonstrated in C. elegans, where injection of dsRNA corresponding to specific genes led to potent and heritable silencing, far more effective than single-stranded RNA. siRNAs play key roles in antiviral defense and transposon suppression, with applications in experimental across eukaryotes. Long non-coding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides, exhibit diverse regulatory functions including modification, transcriptional interference, and post-transcriptional modulation. Many lncRNAs act as scaffolds for protein complexes, recruiting histone modifiers like Polycomb repressive complex 2 (PRC2) to specific genomic loci. A prominent example is , a 17-19 kb lncRNA essential for X-chromosome inactivation in female mammals, where it coats the inactive in cis, leading to epigenetic silencing through recruitment of silencing factors and compaction. Another well-studied lncRNA, HOTAIR (HOX transcript antisense RNA), is a 2.2 kb transcript from the HOXC locus that represses HOXD genes in trans by interacting with PRC2 and LSD1 to promote H3K27 methylation and H3K4 demethylation, respectively, thereby establishing repressive domains. lncRNAs like these are implicated in developmental patterning and cancer progression when dysregulated. Enhancer RNAs (eRNAs) are short, often bidirectional ncRNAs transcribed from enhancer regions, typically 50-2000 long, that facilitate enhancer-promoter interactions to activate transcription. eRNAs promote by stabilizing loops, recruiting and complexes, or interacting with transcription factors like YY1 to enhance activity at target promoters. Their discovery stemmed from genome-wide mapping of nascent transcripts, revealing pervasive enhancer transcription in active cell states. For example, eRNAs from the β-globin locus enhancers loop to interact with the promoter, boosting expression during . eRNA levels correlate with enhancer activity, providing a dynamic readout of regulatory potential. Piwi-interacting RNAs (piRNAs), 24-31 nucleotides long, form complexes with proteins to silence transposons primarily in cells, protecting integrity from mutagenic insertions. Unlike miRNAs and siRNAs, piRNAs are generated from long single-stranded precursors via a Dicer-independent pathway involving endonuclease, and they exhibit a bias for at the 5' end. In animals, piRNAs guide to transposon loci, inducing formation through H3K9 methylation or transcriptional repression. The ping-pong amplification cycle, where primary piRNAs direct cleavage of sense transcripts to produce secondary piRNAs, amplifies the response. Seminal studies in mice identified piRNAs bound to MIWI and MILI, clustered in germline-specific loci, underscoring their role in and transposon control.

Structural and Catalytic RNAs

Structural and catalytic RNAs encompass a diverse class of non-coding RNAs that provide essential scaffold and enzymatic functions within the cell, most prominently in the machinery and RNA processing pathways. These molecules, including ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), form the core structural components of the , facilitating protein synthesis, while ribozymes demonstrate RNA's capacity for independent of proteins. Unlike protein-coding or regulatory RNAs, structural and catalytic RNAs primarily enable constitutive cellular processes through their architectural and reactive properties. Ribosomal RNA (rRNA) constitutes the majority of the ribosome's mass and serves as its structural and functional backbone. In prokaryotes, the small ribosomal subunit contains 16S rRNA, while the large subunit includes 23S and 5S rRNAs; in eukaryotes, these correspond to 18S rRNA in the small subunit and 28S, 5.8S, and 5S rRNAs in the large subunit. These rRNAs fold into complex three-dimensional structures that position ribosomal proteins and create functional sites for . A highly conserved core region within the 23S/28S rRNA forms the peptidyl transferase center (PTC), the site responsible for catalyzing formation during protein synthesis. Transfer RNA (tRNA) molecules act as adapters , linking to their corresponding codons on through specific . The canonical tRNA secondary adopts a cloverleaf conformation, characterized by an acceptor stem, D-, anticodon , and T-, which folds into an L-shaped tertiary . The anticodon loop, located at one end of the L-shape, contains a three-nucleotide anticodon sequence that base-pairs with mRNA codons to ensure accurate selection. At the opposite end, the 3' CCA terminus serves as the attachment site for the , a process catalyzed by aminoacyl-tRNA synthetases that recognize specific tRNA identity elements to achieve high-fidelity charging. Ribozymes represent RNA molecules with intrinsic catalytic activity, exemplified by self-splicing introns and RNase P. Group I self-splicing introns excise themselves from precursor RNAs using a nucleotide or its derivatives as a cofactor, initiating reactions that join the flanking exons without protein assistance. In contrast, undergo self-splicing via two steps, resulting in a lariat intermediate where the intron's 5' end branches to a bulged , mirroring the mechanism of spliceosomal introns. RNase P, a ribonucleoprotein complex, processes the 5' leader sequence of precursor tRNAs to generate mature tRNAs; its RNA subunit alone exhibits catalytic activity , cleaving pre-tRNA substrates in the presence of monovalent and divalent cations. The itself functions as a , with its peptidyl transferase activity residing entirely within the rRNA component of the large subunit. Biochemical and structural studies have shown that the PTC, composed of rRNA without direct involvement of ribosomal proteins, catalyzes the nucleophilic attack of the aminoacyl-tRNA's alpha-amino group on the peptidyl-tRNA's ester linkage to form a . This RNA-based underscores the ancient evolutionary origins of the , predating protein synthesis machinery. Post-transcriptional modifications enhance the stability and functionality of structural RNAs like tRNAs and rRNAs. In tRNAs, hypermodifications such as wybutosine at position 37 (adjacent to the anticodon) stabilize the codon-anticodon interaction through base-stacking, preventing frameshifting and promoting efficient fidelity. Similar modifications in rRNAs, including pseudouridylation and 2'-O-methylation in the PTC and decoding regions, fine-tune ribosomal and catalytic .

Genetic and Evolutionary Roles

RNA Genomes and Viruses

RNA serves as the hereditary material in numerous viruses, where it functions as the genome rather than DNA, enabling rapid replication and evolution in host cells. This contrasts with the DNA-based genomes of cellular organisms and most organelles, highlighting RNA's versatility in genetic systems. The Baltimore classification system categorizes viruses into seven groups based on their genome type (DNA or RNA, single- or double-stranded, sense or antisense) and replication strategy, with groups III through VI encompassing those with RNA genomes. Developed in 1971, this framework underscores how RNA viruses exploit host machinery while relying on virus-encoded polymerases for genome replication, often leading to high genetic diversity. Positive-sense single-stranded RNA (+ssRNA) viruses, classified in group IV, possess genomes that directly serve as (mRNA) upon entry into host cells, allowing immediate of viral proteins including (RdRp). For example, (a ) uses its ~7.5 kb +ssRNA genome to produce RdRp, which then synthesizes a complementary negative-sense strand as a template for new +ssRNA genomes, facilitating efficient replication in cytoplasmic membrane-bound compartments. Negative-sense single-stranded RNA (-ssRNA) viruses, in group V, carry their genome in an antisense orientation and package RdRp within the virion to first transcribe positive-sense mRNAs for protein synthesis before full genome replication can occur. , with its segmented ~13.5 kb -ssRNA genome, exemplifies this: the viral complex initiates transcription in the nucleus, producing mRNAs capped by host factors, which are translated to support subsequent replication of full-length antigenomic intermediates. Double-stranded RNA (dsRNA) viruses, grouped in class III, feature genomes composed of 10–12 linear segments encapsidated within the virion, providing stability against host nucleases. Reoviruses, such as mammalian orthoreovirus, have a ~24 kb dsRNA divided into large, medium, and small segments, each encoding specific proteins; replication occurs in cytoplasmic viral factories where endogenous RdRp transcribes and replicates segments without free RNA intermediates, enabling genetic reassortment during co-infection. Retroviruses, in group VI, maintain single-stranded RNA genomes (~9 kb) that are reverse-transcribed into DNA , which integrate into the host as stable hereditary elements. Human immunodeficiency virus (HIV-1), for instance, forms a double-stranded DNA via its RNA template, allowing persistent infection and propagation with host DNA during . A key challenge in RNA virus replication stems from the error-prone nature of RdRp enzymes, which lack proofreading mechanisms found in DNA polymerases, resulting in mutation rates of approximately 10^{-4} to 10^{-5} errors per nucleotide per replication cycle—orders of magnitude higher than DNA-based systems. This quasispecies diversity drives rapid viral evolution, immune evasion, and adaptation to antiviral therapies, but also imposes fitness costs on progeny virions. In organelles like mitochondria and chloroplasts, genetic material is primarily circular DNA (e.g., ~16 kb mitochondrial DNA in humans), yet in animal cells, RNA transcripts from mitochondrial genomes can constitute a significant portion (up to 30%) of the cellular mRNA pool in metabolically active tissues like heart muscle, supporting organelle function and indirectly influencing hereditary traits through post-transcriptional regulation. Evolutionarily, RNA genomes in viruses suggest ancient origins, potentially mirroring primordial RNA-world scenarios, and their high mutability facilitates diversification across host species, contributing to zoonotic emergences.

Reverse Transcription and Retroelements

Reverse transcription is the process by which DNA is synthesized from an RNA template, a mechanism first discovered in 1970 by Howard Temin and Satoshi Mizutani, who identified an RNA-dependent in virions of , and independently by in RNA tumor viruses, challenging the . This enzyme, known as (RT), was recognized for its role in retroviral replication, earning Temin and Baltimore the 1975 in or . Reverse transcriptase is a multifunctional encoded by the pol gene in retroviruses, such as HIV-1, where it is produced as part of the Gag-Pol polyprotein and exhibits both activity for RNA-templated synthesis and RNase H activity to degrade the RNA strand in RNA-DNA hybrids. In HIV-1, RT forms a heterodimer consisting of p66 (catalytic subunit) and p51 (structural subunit), with the polymerase domain in p66 responsible for nucleotide addition and the RNase H domain cleaving RNA in hybrid duplexes. The enzyme's error-prone nature, with a rate of approximately 1 error per 10,000 incorporated, contributes to high mutation rates in retroviral genomes, driving viral diversity and . The mechanism of reverse transcription begins upon retroviral entry into the host cell, where RT uses a tRNA primer annealed to the viral RNA genome's primer to synthesize a strand, forming an RNA-DNA hybrid. RNase H activity then partially degrades the RNA template, allowing the newly synthesized DNA to fold back via repeat sequences to prime second-strand synthesis, ultimately yielding a double-stranded DNA (dsDNA) flanked by long terminal repeats (LTRs) generated through template switching during the process. These LTRs, identical direct repeats at both ends of the proviral DNA, contain promoter and enhancer elements essential for viral gene expression after integration into the host genome. Retroelements are that propagate via RNA intermediates and reverse transcription, comprising a significant portion of eukaryotic genomes. Endogenous retroviruses (ERVs), ancient integrations of retroviral proviruses, account for about 8% of the , with many retaining LTRs but lacking functional gag, pol, and env genes due to over evolutionary time. Non-LTR retrotransposons include long interspersed nuclear elements (LINEs), such as LINE-1, which encode their own RT and are autonomously mobile, and short interspersed nuclear elements (SINEs), like Alu elements, which are non-autonomous and rely on LINE-1 machinery for retrotransposition via RNA intermediates. Alu elements, the most abundant in with over one million copies, amplify through transcription into RNA, reverse transcription, and reintegration, influencing genome structure and sometimes contributing to genetic disorders. Telomerase represents a cellular application of reverse transcription, consisting of the (TERT) protein subunit with RT activity and the telomerase RNA component (TERC), which provides the template sequence for adding telomeric repeats to ends. In humans, TERC's 3'-CAC-5' template directs TERT to extend the 3' overhang of telomeres by adding TTAGGG repeats, counteracting replicative shortening and maintaining genomic stability in stem and cancer cells. This ribonucleoprotein complex exemplifies how reverse transcription mechanisms, originally identified in viruses, are co-opted for essential eukaryotic functions.

Double-Stranded and Circular RNAs

Double-stranded RNA (dsRNA) serves as a critical intermediate in the replication of many RNA viruses, where it is generated by viral RNA-dependent RNA polymerases (RdRps) during the synthesis of complementary strands from positive-sense RNA templates. This biogenesis process is essential for viral genome amplification, as RdRps use the dsRNA as a template to produce progeny genomic RNA. In cellular contexts, dsRNA also arises during RNA interference (RNAi) pathways, where double-stranded precursors are processed into small interfering RNAs (siRNAs) to silence gene expression. dsRNA plays a pivotal role in triggering innate immune responses, primarily through activation of the protein kinase R (PKR) pathway, which phosphorylates 2α (eIF2α) to inhibit global protein translation and induce stress responses. This activation leads to the production of type I interferons (IFNs) via downstream signaling, enhancing antiviral defenses by upregulating interferon-stimulated genes. Additionally, dsRNA serves as a trigger for RNAi-mediated antiviral immunity, where enzymes cleave it into siRNAs that guide proteins to degrade viral RNA. In viruses with RNA genomes, these dsRNA intermediates are key targets for host recognition, linking them to broader antiviral mechanisms. Evolutionarily, dsRNA structures are conserved in innate immune pathways across eukaryotes, suggesting ancient origins in antiviral defense. Circular RNAs (circRNAs) are a class of non-coding RNAs formed through back-splicing, a process where a downstream splice donor joins an upstream splice acceptor, often facilitated by complementary sequences in flanking introns or . Biogenesis can involve lariat intermediates from canonical splicing, where introns are removed and the lariat's branch point enables ligation to form the circular structure. This covalent closure renders circRNAs highly resistant to degradation, conferring greater stability compared to linear RNAs. circRNAs exert regulatory functions, notably as microRNA (miRNA) sponges that sequester miRNAs and prevent their interaction with target mRNAs; a prominent example is ciRS-7 (also known as CDR1as), which harbors over 70 binding sites for miR-7 and modulates neuronal . In select cases, circRNAs undergo translation to produce proteins, particularly when containing internal ribosome entry sites (IRES) or undergoing cap-independent initiation, as observed in some viral and cellular circRNAs. Detection of circRNAs typically involves enrichment with RNase R, an that digests linear RNAs but spares circular forms, followed by RNA sequencing to identify back-spliced junctions. In certain cell types, such as neurons, circRNAs can comprise 10-20% of the , reflecting their high abundance and stability. Recent studies since 2020 have highlighted circRNAs' involvement in neurodegeneration, where dysregulated circRNAs in Alzheimer's and Parkinson's diseases influence synaptic function and amyloid-beta accumulation through miRNA sponging and protein interactions. For dsRNA, post-2020 research has elucidated its role in enhancing antiviral transcriptional responses independent of sequence-specific recognition, bolstering innate immunity against emerging pathogens like SARS-CoV-2. Evolutionarily, circular RNAs, emerging from , represent an evolutionary innovation in higher organisms, with over 100,000 identified in humans, potentially enhancing regulatory diversity without genomic expansion.

Historical and Fundamental Discoveries

Early Identification and Characterization

In 1869, Swiss biochemist Friedrich Miescher isolated a phosphorus-rich substance he termed "nuclein" from the nuclei of white blood cells obtained from discarded surgical bandages, marking the first identification of nucleic acids, which include both DNA and RNA. Miescher's extraction involved treating the cells with pepsin to remove proteins, followed by alkali to precipitate the nuclein, revealing its acidic nature and high phosphorus content, distinct from known proteins or lipids. Although Miescher did not distinguish between DNA and RNA at the time, his work laid the groundwork for recognizing nucleic acids as fundamental cellular components. The identification of RNA as a distinct emerged in the late 1930s through studies on the (TMV). In 1936, Roy Markham and Northrop demonstrated the presence of a in purified TMV preparations, and by 1937, Bawden and Pirie confirmed it contained , indicating a composition. Further analysis in 1939 by Bawden and Pirie established that the was ribonucleic acid (RNA), not deoxyribonucleic acid, based on its susceptibility to hydrolysis and base composition. Wendell Stanley, who had crystallized TMV in 1935, collaborated with Bawden and Pirie, and by 1944, their collective work proposed RNA's potential role in viral heredity, challenging the protein-centric views of inheritance prevalent at the time. In the , the emerging field of began elucidating RNA's functional roles in protein synthesis. proposed the "adaptor hypothesis" in 1955, suggesting that molecules act as intermediaries to translate the sequences of a genetic template into chains, addressing the mismatch between the four-letter code and the twenty . This idea, detailed in Crick's 1958 paper "On Protein Synthesis," posited that these adaptors—later identified as transfer RNAs (tRNAs)—recognize specific codons via base-pairing while carrying attached , thus serving as the bridge in the . The hypothesis provided a conceptual framework for RNA's intermediary function, influencing subsequent experiments on genetic coding. The concept of messenger RNA (mRNA) was experimentally validated in 1961 through studies by Sydney Brenner, François Jacob, and Matthew Meselson using T4 bacteriophage infection in Escherichia coli. Their pulse-labeling experiments with radioactive uracil demonstrated the existence of a short-lived, unstable RNA species that rapidly incorporates genetic information from newly synthesized DNA and directs protein synthesis at ribosomes. Published in Nature as "An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis," the work showed that this RNA turns over quickly, with a half-life of about 2-3 minutes, confirming its role as a transient messenger between DNA and ribosomes. This discovery resolved debates about how genetic information flows in bacteria and established mRNA as the key intermediary in gene expression. The 1970s brought structural insights into RNA components and the revelation of gene organization complexities. In 1974, Alexander Rich and Sung-Hou Kim, along with colleagues, determined the three-dimensional crystal structure of yeast phenylalanine tRNA at 3.0 Å resolution, revealing its L-shaped tertiary fold with stacked helices and a cloverleaf secondary structure stabilized by modified bases and magnesium ions. This structure, resolved using diffraction on crystals grown from purified tRNA, confirmed Crick's adaptor by showing the anticodon loop positioned to interact with mRNA and the acceptor stem for attachment. Concurrently, in 1977, Phillip Sharp and Richard Roberts independently discovered introns—non-coding sequences interrupting eukaryotic s—through electron microscopy of adenovirus RNA hybrids, revealing looped-out regions where introns are transcribed but spliced out during mRNA maturation. Sharp's team at MIT and Roberts' at Cold Spring Harbor used heteroduplex mapping to show that the beta-globin and s contain intervening sequences, fundamentally altering views of gene continuity. These findings, awarded the 1993 in Physiology or Medicine, highlighted RNA's role in post-transcriptional processing.

Milestones in RNA Function and Regulation

In 1982, Thomas Cech's laboratory discovered the self-splicing capability of the ribosomal RNA intron from Tetrahymena thermophila, demonstrating that RNA could catalyze its own excision without protein assistance, thus identifying the first ribozyme. This breakthrough challenged the prevailing view that only proteins function as enzymes and paved the way for understanding RNA's catalytic potential. Independently, in 1983, Sidney Altman's group showed that the RNA component of RNase P from Escherichia coli performs the catalytic cleavage of tRNA precursors, confirming RNA's enzymatic role in vivo. Their discoveries, recognized with the 1989 Nobel Prize in Chemistry, established ribozymes as key players in RNA processing and regulation, influencing fields from splicing mechanisms to synthetic biology. The late 1990s brought further revelations in RNA-mediated gene silencing with the 1998 identification of RNA interference (RNAi) by Andrew Fire and Craig Mello, who demonstrated that double-stranded RNA triggers potent, sequence-specific degradation of homologous mRNAs in Caenorhabditis elegans. This work elucidated the core RNAi pathway involving small interfering RNAs (siRNAs) and microRNAs (miRNAs), which guide Argonaute proteins to target transcripts for cleavage or translational repression, thereby regulating gene expression at the post-transcriptional level. Awarded the 2006 Nobel Prize in Physiology or Medicine, RNAi revolutionized functional genomics, enabling targeted gene knockdown and inspiring therapeutic applications like siRNA drugs for viral infections and genetic disorders. The and marked an explosion in recognizing regulatory non-coding RNAs, particularly long non-coding RNAs (lncRNAs), with early examples like —first sequenced in 1991 but functionally characterized in the mid- for its role in X-chromosome inactivation through coating and silencing. By 2012, the project's GENCODE consortium cataloged over 9,000 human lncRNA loci, revealing their widespread expression and diverse regulatory functions, such as epigenetic modulation and transcriptional interference, far beyond initial annotations. This systematic annotation, building on computational pipelines from the late , highlighted lncRNAs' prevalence—comprising up to 80% of the non-coding —and spurred genome-wide studies into their roles in development and disease. Advancements in epitranscriptomics emerged in the 2010s, with Kate D. Meyer's 2012 development of MeRIP-seq enabling transcriptome-wide mapping of N⁶-methyladenosine (m⁶A), the most abundant internal mRNA modification, enriched near stop codons and in 3' UTRs to influence splicing, stability, and translation. Concurrently, Julia Salzman's 2012 analysis uncovered circular RNAs (circRNAs) as predominant isoforms from thousands of human genes, formed via back-splicing and functioning as miRNA sponges or regulators of parental , challenging linear RNA paradigms. These discoveries expanded RNA to include chemical modifications and topologies, with m⁶A "writers" like METTL3 and circRNA abundance in neural tissues underscoring their tissue-specific impacts. From 2020 onward, research has illuminated RNA's role in biomolecular condensates, particularly within stress granules—cytoplasmic assemblies that sequester mRNAs during cellular stress to halt and promote . A pivotal 2020 study revealed G3BP1 as a core driver, where RNA binding induces its conformational switch to trigger liquid-liquid , dynamically partitioning RNAs for selective protection or degradation. Single-cell RNA sequencing (scRNA-seq) has further resolved regulatory networks, with tools like IReNA (2022) integrating scRNA-seq and scATAC-seq to infer cell-type-specific interactions, uncovering dynamic modules in heterogeneous tissues like tumors. The accelerated RNA research through mRNA vaccines, which by 2021 demonstrated scalable production and immune efficacy, spurring over 200 clinical trials for non-viral applications like cancer immunotherapies and boosting lipid nanoparticle delivery innovations. These developments, from 2020 to 2025, have integrated phase-separated RNA dynamics with high-resolution profiling, transforming regulatory insights and therapeutic pipelines.

RNA in Abiogenesis and Prebiotic Chemistry

The RNA world hypothesis posits that RNA served as both the genetic material and catalyst in the earliest stages of life on Earth, preceding the emergence of DNA and proteins. Proposed by Walter Gilbert in 1986, this model suggests that self-replicating RNA molecules capable of catalyzing their own replication and basic metabolic reactions formed the foundation of prebiotic evolution. In this scenario, RNA's dual functionality—storing information like DNA and performing enzymatic roles like proteins—allowed it to bootstrap the complexity of life without requiring more sophisticated biopolymers initially. Prebiotic synthesis pathways for RNA components remain a central focus, with research exploring plausible geochemical environments. , the building blocks of RNA, could have formed in settings such as formamide-rich pools or hydrothermal vents, where simple precursors like and react under mild conditions to yield sugars and nucleobases. A landmark achievement came in , when Matthew Powner and colleagues demonstrated the synthesis of ribonucleotides—such as and derivatives—from simple prebiotic molecules like , , and , bypassing the unstable free and requiring only wet-dry cycles for activation. This pathway, conducted under conditions mimicking , produced activated with 3'-5' phosphodiester linkages, addressing a key hurdle in RNA . Hydrothermal vents provide another proposed site, where surfaces catalyze assembly from CO2 and H2 under high-temperature gradients. Despite these advances, significant challenges persist in reconstructing a fully RNA-based prebiotic . One major issue is the preferential formation of non-standard 2'-5' phosphodiester linkages during non-enzymatic , which destabilize RNA duplexes and hinder template-directed replication compared to the biologically relevant 3'-5' bonds. These aberrant linkages arise because prebiotic reactions often activate the 2'-hydroxyl group on , leading to branched polymers that are less stable and prone to . experiments, such as Sol Spiegelman's 1967 work with Qβ phage RNA replicase, illustrate the dynamics of RNA simplification under selective pressure; serial transfer in test tubes produced "Spiegelman's monster," a truncated 218-nucleotide RNA that replicated rapidly but lost non-essential genetic information, highlighting the ease of evolutionary regression without stabilizing mechanisms. Supporting evidence for the RNA world includes ribozymes that mimic primitive metabolic functions, demonstrating RNA's catalytic versatility. For instance, in vitro-selected ribozymes have been engineered to perform reactions akin to glycolysis intermediates, such as carbon-carbon bond formation, suggesting that early RNA networks could sustain basic without proteins. Extraterrestrial delivery of RNA precursors further bolsters the hypothesis; the , which fell in 1969, contains a suite of nucleobases including , , , uracil, and , with isotopic signatures indicating abiotic synthesis in space. These compounds, detected at concentrations up to 70 , could have seeded Earth's prebiotic soups with ready-made building blocks. Recent computational and experimental studies from 2023 to 2025 have refined models of prebiotic RNA pathways using and co-evolution simulations. In 2024, studies on vesicle-RNA co-evolution demonstrated that vesicles encapsulate short RNA oligomers, enhancing their stability and enabling template-directed ligation in dilute prebiotic conditions, with encapsulated RNAs showing up to 10-fold faster replication rates compared to free molecules. These models suggest that membranes and RNA co-emerged, facilitating the transition from abiotic chemistry to Darwinian . , as seen in modern structural RNAs, provides a brief analog for such ancient functions, where self-splicing introns hint at primordial RNA processing capabilities.

Applications in Medicine and Biotechnology

Therapeutic RNA Molecules

Therapeutic RNA molecules represent a rapidly advancing class of pharmaceuticals that leverage RNA's natural roles in and regulation to treat diseases. These include messenger RNA (mRNA) vaccines, antisense oligonucleotides (), small interfering RNAs (siRNAs), and aptamers, each designed to modulate specific biological processes such as protein production, , or protein binding. Unlike traditional small-molecule drugs, offer high specificity and the potential for rapid development, particularly in response to emerging threats like infectious diseases or genetic disorders. Their clinical success has been enabled by innovations in chemical modifications and delivery systems to overcome inherent RNA vulnerabilities. mRNA vaccines, a breakthrough in prophylactic and therapeutic applications, instruct host cells to produce antigenic proteins that trigger immune responses. The Pfizer-BioNTech vaccine (BNT162b2), approved by the U.S. (FDA) in December 2020 under , and the Moderna vaccine (mRNA-1273), authorized shortly thereafter, both encode the within nucleoside-modified mRNA encapsulated in nanoparticles (LNPs) for efficient cellular uptake and from degradation. Upon delivery, the mRNA is translated by ribosomes into the , eliciting neutralizing antibodies and T-cell immunity without using live virus. These vaccines demonstrated over 90% efficacy in preventing symptomatic in phase 3 trials, marking the first widespread deployment of mRNA technology in humans. Antisense oligonucleotides (ASOs) function by hybridizing to target RNA sequences to alter splicing, block , or induce degradation, providing precise control over . Nusinersen (Spinraza), an ASO approved by the FDA in December 2016 for (SMA), binds to an intronic splicing silencer site in SMN2 pre-mRNA, promoting inclusion of 7 to increase full-length survival (SMN) protein production. Administered intrathecally, it has shown significant improvements in motor function for infants and children with SMA in clinical trials, with sustained benefits observed over multiple years. Similarly, siRNA therapeutics exploit to silence disease-causing genes. Patisiran (Onpattro), approved by the FDA in August 2018 for hereditary transthyretin-mediated (hATTR) , is a lipid nanoparticle-formulated siRNA conjugated to (GalNAc) for hepatocyte-specific targeting via the . It reduces hepatic (TTR) production by over 80% in patients, alleviating symptoms as evidenced in the APOLLO phase 3 trial. Aptamers, single-stranded RNA or DNA ligands selected for high-affinity binding to target proteins, offer a non-immunogenic alternative for inhibiting protein function. Pegaptanib (Macugen), the first FDA-approved aptamer in December 2004 for neovascular (wet) age-related macular degeneration (AMD), is a 27-nucleotide RNA molecule pegylated for stability that specifically binds the vascular endothelial growth factor (VEGF165) isoform, preventing its interaction with receptors and reducing pathological angiogenesis in the retina. Intravitreal injections slowed vision loss in about 70% of treated patients in pivotal trials, establishing aptamers as viable therapeutics despite later competition from protein-based anti-VEGFs. Key challenges in RNA therapeutics include rapid enzymatic degradation and innate immune activation, which can limit efficacy and cause adverse reactions. To enhance stability, modifications such as (Ψ) are incorporated, as in mRNA vaccines, where Ψ substitution reduces recognition, lowers immunogenicity, and boosts translation efficiency by up to 10-fold compared to unmodified RNA. LNPs and GalNAc conjugates further address delivery barriers by facilitating endosomal escape and tissue-specific uptake, though off-target effects and manufacturing scalability remain hurdles. As of 2025, mRNA platforms are expanding into , with BioNTech's individualized neoantigen-specific mRNA vaccines (e.g., autogene cevumeran) showing promising immune activation and tumor reduction in phase 2 trials for pancreatic and cancers, with phase 3 studies planned or initiated in 2025. Recent approvals include donidalorsen in August 2025 for , further expanding the portfolio of RNA-based treatments. These developments underscore RNA's potential for , with over a dozen RNA drugs now FDA-approved.

Diagnostic and Research Tools

(RT-PCR) and quantitative PCR (qPCR) are foundational techniques for RNA detection in diagnostics, involving the conversion of RNA to followed by amplification to quantify viral or levels. These methods gained prominence during the for detection, where RT-qPCR served as the gold standard due to its high sensitivity (detecting as few as 10-100 viral RNA copies) and specificity exceeding 99%, enabling rapid identification of infected individuals from nasopharyngeal swabs. Limitations include potential false negatives from low viral loads or sample degradation, but optimizations like one-step RT-qPCR have improved throughput for large-scale testing. RNA sequencing () represents a high-throughput approach for comprehensive analysis, capturing the full spectrum of RNA molecules to profile , , and novel transcripts in research and disease diagnostics. By sequencing cDNA libraries from RNA samples, RNA-seq provides quantitative data on thousands of genes simultaneously, outperforming earlier methods in and resolution, with applications in identifying biomarkers for cancers like through differential expression patterns. Single-cell RNA-seq (scRNA-seq) extends this by resolving cellular heterogeneity, isolating transcripts from individual cells to map rare subpopulations, such as tumor-infiltrating immune cells, which bulk methods obscure; for instance, scRNA-seq has revealed subtype-specific gene signatures in heterogeneity with over 10,000 cells profiled per sample. Microarrays and Northern blots offer targeted tools for RNA expression profiling, though they have been largely supplanted by sequencing in modern workflows. DNA microarrays hybridize labeled RNA or cDNA to immobilized probes on a chip, enabling parallel assessment of up to 50,000 genes to detect expression changes, as validated in studies of inflammatory responses where fold-changes correlated with clinical outcomes. Northern blots, a classical gel-based method, separate RNA by size via , transfer it to a , and detect specific transcripts using radiolabeled or chemiluminescent probes, providing size confirmation and quantification for validation, such as confirming levels in developmental tissues with sensitivity down to 1-5 pg of target RNA. In situ hybridization (ISH) enables spatial visualization of RNA localization within intact tissues, using labeled probes to bind target sequences and reveal expression patterns at cellular resolution. RNA-ISH, often fluorescent (FISH), has been pivotal in for mapping mRNA distribution in sections, identifying localized transcripts like those for neurotransmitters with single-molecule sensitivity, and in for diagnosing viral infections or oncogenic fusions in tumor biopsies. Advances like branched DNA amplification in ISH platforms enhance signal detection in formalin-fixed tissues, achieving multiplexed analysis of up to 48 RNA targets simultaneously. Spatial transcriptomics, exemplified by the Visium platform, integrates with tissue imaging to map at near-single-cell resolution, addressing limitations of dissociated samples by preserving spatial context. Introduced in 2019 and refined by 2024 with high-definition versions capturing 2-micron pixels across 1 cm² sections, Visium has elucidated tumor microenvironments in , identifying spatially segregated immune niches with over 18,000 genes profiled per spot. These methods support research into tissue architecture in diseases like , where zonal gene gradients inform . Post-2023 integrations of (AI) with RNA diagnostics have enhanced analysis of complex datasets, such as using on RNA-seq outputs for predictive modeling in . AI algorithms, like deep neural networks trained on scRNA-seq data, classify cancer subtypes with 95% accuracy by detecting subtle expression patterns missed by traditional statistics, as demonstrated in pancreatic ductal adenocarcinoma diagnostics. In viral diagnostics, AI-optimized RT-qPCR interpretation reduces false positives by 20% through in amplification curves, accelerating outbreak responses.

Emerging Synthetic and Editing Technologies

Emerging advancements in RNA synthetic and editing technologies have revolutionized and by enabling precise manipulation of genetic material at the RNA level. The CRISPR-Cas systems, originally derived from bacterial immune defenses, utilize single-guide RNAs (sgRNAs) to direct nucleases for targeted DNA cleavage and editing, allowing for insertions, deletions, or base substitutions in eukaryotic genomes. In parallel, Cas13 variants target RNA directly, facilitating transient knockdown or cleavage without altering the underlying DNA sequence, which is particularly useful for studying function or degrading viral RNAs. RNA editing technologies have advanced through the integration of components with endogenous enzymes like , which naturally catalyze adenosine-to-inosine (A-to-I) in RNA transcripts. For instance, CRISPRoff employs a catalytically dead fused to a KRAB repressor domain to achieve epigenetic silencing of DNA targets via , offering reversible repression without permanent mutations.31502-6) Building on ADAR's mechanism, the REPAIR system, developed in 2017, uses a catalytically inactive Cas13 fused to an evolved ADAR deaminase to enable programmable A-to-I edits in target RNAs, demonstrating up to 30% editing efficiency in cellular transcripts with minimal off-target effects. In , RNA molecules serve as programmable building blocks for regulatory circuits and nanostructures. Toehold switches, short RNA sequences that form metastable hairpins, act as riboregulators by activating translation upon binding complementary trigger RNAs, enabling logic-gated in and mammalian cells with sensitivities rivaling protein-based sensors.00864-0) RNA nanostructures, such as those created via RNA techniques, fold into complex two- and three-dimensional shapes through computational design of base-pairing motifs, achieving nanoscale assemblies stable under physiological conditions for potential use in . Aptamer evolution through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process generates high-affinity RNA ligands that bind specific targets, such as proteins or small molecules, with dissociation constants in the nanomolar range. For vaccine development, mRNA incorporates modifications like to reduce and enhance stability, as exemplified in the rapid deployment of mRNA vaccines that elicited robust immune responses in clinical trials. As of 2025, frontiers in emphasize self-assembling RNA particles for , where lipid-RNA nanoparticles encapsulate therapeutics to improve and reduce systemic toxicity in cancer therapies. Recent Cas13-based antiviral trials in 2024 demonstrated prophylactic efficacy against in animal models by degrading viral RNA , paving the way for RNA-targeted antivirals. Additionally, quantum dot-RNA hybrid sensors have emerged for real-time detection of RNA biomarkers, leveraging energy transfer to achieve single-molecule sensitivity in diagnostic applications.

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