Ribosome

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Ribosome 

Large (red) and small (blue) subunits of a ribosome

Cell biology
Animal cell diagram
Components of a typical animal cell: Nucleolus Nucleus Ribosome (dots as part of 5) Vesicle Rough endoplasmic reticulum Golgi apparatus (or, Golgi body) Cytoskeleton Smooth endoplasmic reticulum Mitochondrion Vacuole Cytosol (fluid that contains organelles; with which, comprises cytoplasm) Lysosome Centrosome Cell membrane

Ribosomes (/ˈraɪbəzoʊm, -soʊm/) are macromolecular biological machines, found within all cells, that perform messenger RNA translation. Ribosomes link amino acids together in the order specified by the codons of messenger RNA molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA molecules and many ribosomal proteins (r-proteins). The ribosomes and associated molecules are also known as the translational apparatus.

Overview

The sequence of DNA that encodes the sequence of the amino acids in a protein is transcribed into a messenger RNA (mRNA) chain. Ribosomes bind to the messenger RNA molecules and use the RNA's sequence of nucleotides to determine the sequence of amino acids needed to generate a protein. Amino acids are selected and carried to the ribosome by transfer RNA (tRNA) molecules, which enter the ribosome and bind to the messenger RNA chain via an anticodon stem loop. For each coding triplet (codon) in the messenger RNA, there is a unique transfer RNA that must have the exact anti-codon match, and carries the correct amino acid for incorporating into a growing polypeptide chain. Once the protein is produced, it can then fold to produce a functional three-dimensional structure.

A ribosome is made from complexes of RNAs and proteins and is therefore a ribonucleoprotein complex. In prokaryotes each ribosome is composed of small (30S) and large (50S) components, called subunits, which are bound to each other:

1. (30S) has mainly a decoding function and is also bound to the mRNA
2. (50S) has mainly a catalytic function and is also bound to the aminoacylated tRNAs.

The synthesis of proteins from their building blocks takes place in four phases: initiation, elongation, termination, and recycling. The start codon in all mRNA molecules has the sequence AUG. The stop codon is one of UAA, UAG, or UGA; since there are no tRNA molecules that recognize these codons, the ribosome recognizes that translation is complete. When a ribosome finishes reading an mRNA molecule, the two subunits separate and are usually broken up but can be reused. Ribosomes are a kind of enzyme, called ribozymes because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA.

In eukaryotic cells, ribosomes are often associated with the intracellular membranes that make up the rough endoplasmic reticulum.

Ribosomes from bacteria, archaea, and eukaryotes (in the three-domain system) resemble each other to a remarkable degree, evidence of a common origin. They differ in their size, sequence, structure, and the ratio of protein to RNA. The differences in structure allow some antibiotics to kill bacteria by inhibiting their ribosomes while leaving human ribosomes unaffected. In all species, more than one ribosome may move along a single mRNA chain at one time (as a polysome), each "reading" a specific sequence and producing a corresponding protein molecule.

The mitochondrial ribosomes of eukaryotic cells are distinct from their other ribosomes. They functionally resemble those in bacteria, reflecting the evolutionary origin of mitochondria as endosymbiotic bacteria.

Discovery

Ribosomes were first observed in the mid-1950s by Romanian-American cell biologist George Emil Palade, using an electron microscope, as dense particles or granules. They were initially called Palade granules due to their granular structure. The term "ribosome" was proposed in 1958 by Howard M. Dintzis:

During the course of the symposium a semantic difficulty became apparent. To some of the participants, "microsomes" mean the ribonucleoprotein particles of the microsome fraction contaminated by other protein and lipid material; to others, the microsomes consist of protein and lipid contaminated by particles. The phrase "microsomal particles" does not seem adequate, and "ribonucleoprotein particles of the microsome fraction" is much too awkward. During the meeting, the word "ribosome" was suggested, which has a very satisfactory name and a pleasant sound. The present confusion would be eliminated if "ribosome" were adopted to designate ribonucleoprotein particles in sizes ranging from 35 to 100S.

— Albert Claude, Microsomal Particles and Protein Synthesis

Albert Claude, Christian de Duve, and George Emil Palade were jointly awarded the Nobel Prize in Physiology or Medicine, in 1974, for the discovery of the ribosome. The Nobel Prize in Chemistry 2009 was awarded to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath for determining the detailed structure and mechanism of the ribosome.

Structure

Ribosomes assemble polymeric protein molecules, the order of which is controlled by the messenger RNA's molecule sequence.

Ribosomal RNA composition for prokaryotes and eukaryotes

The ribosome is a complex cellular machine. It is largely made up of specialized RNA known as ribosomal RNA (rRNA) as well as dozens of distinct proteins (the exact number varies slightly between species). The ribosomal proteins and rRNAs are arranged into two distinct ribosomal pieces of different sizes, known generally as the large and small subunits of the ribosome. Ribosomes consist of two subunits that fit together and work as one to translate the mRNA into a polypeptide chain during protein synthesis. Because they are formed from two subunits of non-equal size, they are slightly longer on the axis than in diameter.

Prokaryotic ribosomes

Prokaryotic ribosomes are around 20 nm (200 Å) in diameter and are composed of 65% rRNA and 35% ribosomal proteins. Eukaryotic ribosomes are between 25 and 30 nm (250–300 Å) in diameter with an rRNA-to-protein ratio that is close to 1. Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes do not directly participate in peptide bond formation catalysis, but rather that these proteins act as a scaffold that may enhance the ability of rRNA to synthesize protein.

Molecular structure of the 30S subunit from Thermus thermophilus. Proteins are shown in blue and the single RNA chain in brown.

The ribosomal subunits of prokaryotes and eukaryotes are quite similar.

The unit of measurement used to describe the ribosomal subunits and the rRNA fragments is the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size. This accounts for why fragment names do not add up: for example, bacterial 70S ribosomes are made of 50S and 30S subunits.

Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. E. coli, for example, has a 16S RNA subunit (consisting of 1540 nucleotides) that is bound to 21 proteins. The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins.

Ribosome of E. coli (a bacterium)

ribosome	subunit	rRNAs	r-proteins
70S	50S	23S (2904 nt)	31
		5S (120 nt)
	30S	16S (1542 nt)	21

Affinity label for the tRNA binding sites on the E. coli ribosome allowed the identification of A and P site proteins most likely associated with the peptidyltransferase activity; labelled proteins are L27, L14, L15, L16, L2; at least L27 is located at the donor site, as shown by E. Collatz and A.P. Czernilofsky. Additional research has demonstrated that the S1 and S21 proteins, in association with the 3′-end of 16S ribosomal RNA, are involved in the initiation of translation.

Archaeal ribosomes

Archaeal ribosomes share the same general dimensions of bacteria ones, being a 70S ribosome made up from a 50S large subunit, a 30S small subunit, and containing three rRNA chains. However, on the sequence level, they are much closer to eukaryotic ones than to bacterial ones. Every extra ribosomal protein archaea have compared to bacteria has a eukaryotic counterpart, while no such relation applies between archaea and bacteria.

Eukaryotic ribosomes

Main article: Eukaryotic ribosome

Eukaryotes have 80S ribosomes located in their cytosol, each consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (1900 nucleotides) and 33 proteins. The large subunit is composed of a 5S RNA (120 nucleotides), 28S RNA (4700 nucleotides), a 5.8S RNA (160 nucleotides) subunits and 49 proteins.

eukaryotic cytosolic ribosomes (R. norvegicus)

ribosome	subunit	rRNAs	r-proteins
80S	60S	28S (4718 nt)	49
		5.8S (160 nt)
		5S (120 nt)
	40S	18S (1874 nt)	33

During 1977, Czernilofsky published research that used affinity labeling to identify tRNA-binding sites on rat liver ribosomes. Several proteins, including L32/33, L36, L21, L23, L28/29 and L13 were implicated as being at or near the peptidyl transferase center.

Plastoribosomes and mitoribosomes

Main articles: Mitochondrial ribosome and Chloroplast § Chloroplast ribosomes

In eukaryotes, ribosomes are present in mitochondria (sometimes called mitoribosomes) and in plastids such as chloroplasts (also called plastoribosomes). They also consist of large and small subunits bound together with proteins into one 70S particle. These ribosomes are similar to those of bacteria and these organelles are thought to have originated as symbiotic bacteria. Of the two, chloroplastic ribosomes are closer to bacterial ones than mitochondrial ones are. Many pieces of ribosomal RNA in the mitochondria are shortened, and in the case of 5S rRNA, replaced by other structures in animals and fungi. In particular, Leishmania tarentolae has a minimalized set of mitochondrial rRNA. In contrast, plant mitoribosomes have both extended rRNA and additional proteins as compared to bacteria, in particular, many pentatricopetide repeat proteins.

The cryptomonad and chlorarachniophyte algae may contain a nucleomorph that resembles a vestigial eukaryotic nucleus. Eukaryotic 80S ribosomes may be present in the compartment containing the nucleomorph.

Making use of the differences

The differences between the bacterial and eukaryotic ribosomes are exploited by pharmaceutical chemists to create antibiotics that can destroy a bacterial infection without harming the cells of the infected person. Due to the differences in their structures, the bacterial 70S ribosomes are vulnerable to these antibiotics while the eukaryotic 80S ribosomes are not. Even though mitochondria possess ribosomes similar to the bacterial ones, mitochondria are not affected by these antibiotics because they are surrounded by a double membrane that does not easily admit these antibiotics into the organelle. A noteworthy counterexample is the antineoplastic antibiotic chloramphenicol, which inhibits bacterial 50S and eukaryotic mitochondrial 50S ribosomes. Ribosomes in chloroplasts, however, are different: Antibiotic resistance in chloroplast ribosomal proteins is a trait that has to be introduced as a marker, with genetic engineering.

Common properties

The various ribosomes share a core structure, which is quite similar despite the large differences in size. Much of the RNA is highly organized into various tertiary structural motifs, for example pseudoknots that exhibit coaxial stacking. The extra RNA in the larger ribosomes is in several long continuous insertions, such that they form loops out of the core structure without disrupting or changing it. All of the catalytic activity of the ribosome is carried out by the RNA; the proteins reside on the surface and seem to stabilize the structure.

High-resolution structure

Figure 4: Atomic structure of the 50S subunit from Haloarcula marismortui. Proteins are shown in blue and the two RNA chains in brown and yellow. The small patch of green in the center of the subunit is the active site.

The general molecular structure of the ribosome has been known since the early 1970s. In the early 2000s, the structure has been achieved at high resolutions, of the order of a few ångströms.

The first papers giving the structure of the ribosome at atomic resolution were published almost simultaneously in late 2000. The 50S (large prokaryotic) subunit was determined from the archaeon Haloarcula marismortui and the bacterium Deinococcus radiodurans, and the structure of the 30S subunit was determined from the bacterium Thermus thermophilus. These structural studies were awarded the Nobel Prize in Chemistry in 2009. In May 2001 these coordinates were used to reconstruct the entire T. thermophilus 70S particle at 5.5 Å resolution.

Two papers were published in November 2005 with structures of the Escherichia coli 70S ribosome. The structures of a vacant ribosome were determined at 3.5 Å resolution using X-ray crystallography. Then, two weeks later, a structure based on cryo-electron microscopy was published, which depicts the ribosome at 11–15 Å resolution in the act of passing a newly synthesized protein strand into the protein-conducting channel.

The first atomic structures of the ribosome complexed with tRNA and mRNA molecules were solved by using X-ray crystallography by two groups independently, at 2.8 Å and at 3.7 Å. These structures allow one to see the details of interactions of the Thermus thermophilus ribosome with mRNA and with tRNAs bound at classical ribosomal sites. Interactions of the ribosome with long mRNAs containing Shine-Dalgarno sequences were visualized soon after that at 4.5–5.5 Å resolution.

In 2011, the first complete atomic structure of the eukaryotic 80S ribosome from the yeast Saccharomyces cerevisiae was obtained by crystallography. The model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core. At the same time, the complete model of a eukaryotic 40S ribosomal structure in Tetrahymena thermophila was published and described the structure of the 40S subunit, as well as much about the 40S subunit's interaction with eIF1 during translation initiation. Similarly, the eukaryotic 60S subunit structure was also determined from Tetrahymena thermophila in complex with eIF6.

Function

Ribosomes are minute particles consisting of RNA and associated proteins that function to synthesize proteins. Proteins are needed for many cellular functions, such as repairing damage or directing chemical processes. Ribosomes can be found floating within the cytoplasm or attached to the endoplasmic reticulum. Their main function is to convert genetic code into an amino acid sequence and to build protein polymers from amino acid monomers.

Ribosomes act as catalysts in two extremely important biological processes called peptidyl transfer and peptidyl hydrolysis. The "PT center is responsible for producing protein bonds during protein elongation".

In summary, ribosomes have two main functions: Decoding the message, and the formation of peptide bonds. These two functions reside in the ribosomal subunits. Each subunit is made of one or more rRNAs and many r-proteins. The small subunit (30S in bacteria and archaea, 40S in eukaryotes) has the decoding function, whereas the large subunit (50S in bacteria and archaea, 60S in eukaryotes) catalyzes the formation of peptide bonds, referred to as the peptidyl-transferase activity. The bacterial (and archaeal) small subunit contains the 16S rRNA and 21 r-proteins (Escherichia coli), whereas the eukaryotic small subunit contains the 18S rRNA and 32 r-proteins (Saccharomyces cerevisiae, although the numbers vary between species). The bacterial large subunit contains the 5S and 23S rRNAs and 34 r-proteins (E. coli), with the eukaryotic large subunit containing the 5S, 5.8S, and 25S/28S rRNAs and 46 r-proteins (S. cerevisiae; again, the exact numbers vary between species).

Translation

Main article: Translation (biology)

Ribosomes are the workplaces of protein biosynthesis, the process of translating mRNA into protein. The mRNA comprises a series of codons which are decoded by the ribosome to make the protein. Using the mRNA as a template, the ribosome traverses each codon (3 nucleotides) of the mRNA, pairing it with the appropriate amino acid provided by an aminoacyl-tRNA. Aminoacyl-tRNA contains a complementary anticodon on one end and the appropriate amino acid on the other. For fast and accurate recognition of the appropriate tRNA, the ribosome utilizes large conformational changes (conformational proofreading). The small ribosomal subunit, typically bound to an aminoacyl-tRNA containing the first amino acid methionine, binds to an AUG codon on the mRNA and recruits the large ribosomal subunit. The ribosome contains three RNA binding sites, designated A, P, and E. The A-site binds an aminoacyl-tRNA or termination release factors; the P-site binds a peptidyl-tRNA (a tRNA bound to the poly-peptide chain); and the E-site (exit) binds a free tRNA. Protein synthesis begins at a start codon AUG near the 5' end of the mRNA. mRNA binds to the P site of the ribosome first. The ribosome recognizes the start codon by using the Shine-Dalgarno sequence of the mRNA in prokaryotes and Kozak box in eukaryotes.

Although catalysis of the peptide bond involves the C2 hydroxyl of RNA's P-site adenosine in a proton shuttle mechanism, other steps in protein synthesis (such as translocation) are caused by changes in protein conformations. Since their catalytic core is made of RNA, ribosomes are classified as "ribozymes," and it is thought that they might be remnants of the RNA world.

Figure 5: Translation of mRNA (1) by a ribosome (2)(shown as small and large subunits) into a polypeptide chain (3). The ribosome begins at the start codon of RNA (AUG) and ends at the stop codon (UAG).

In Figure 5, both ribosomal subunits (small and large) assemble at the start codon (towards the 5' end of the mRNA). The ribosome uses tRNA that matches the current codon (triplet) on the mRNA to append an amino acid to the polypeptide chain. This is done for each triplet on the mRNA, while the ribosome moves towards the 3' end of the mRNA. Usually in bacterial cells, several ribosomes are working parallel on a single mRNA, forming what is called a polyribosome or polysome.

Cotranslational folding

The ribosome is known to actively participate in the protein folding. The structures obtained in this way are usually identical to the ones obtained during protein chemical refolding; however, the pathways leading to the final product may be different. In some cases, the ribosome is crucial in obtaining the functional protein form. For example, one of the possible mechanisms of folding of the deeply knotted proteins relies on the ribosome pushing the chain through the attached loop.

Addition of translation-independent amino acids

Presence of a ribosome quality control protein Rqc2 is associated with mRNA-independent protein elongation. This elongation is a result of ribosomal addition (via tRNAs brought by Rqc2) of CAT tails: ribosomes extend the C-terminus of a stalled protein with random, translation-independent sequences of alanines and threonines.

Ribosome locations

Ribosomes are classified as being either "free" or "membrane-bound".

Figure 6: A ribosome translating a protein that is secreted into the endoplasmic reticulum via protein domain dynamics.

Free and membrane-bound ribosomes differ only in their spatial distribution; they are identical in structure. Whether the ribosome exists in a free or membrane-bound state depends on the presence of an ER-targeting signal sequence on the protein being synthesized, so an individual ribosome might be membrane-bound when it is making one protein, but free in the cytosol when it makes another protein.

Ribosomes are sometimes referred to as organelles, but the use of the term organelle is often restricted to describing sub-cellular components that include a phospholipid membrane, which ribosomes, being entirely particulate, do not. For this reason, ribosomes may sometimes be described as "non-membranous organelles".

Free ribosomes

Free ribosomes can move about anywhere in the cytosol, but are excluded from the cell nucleus and other organelles. Proteins that are formed from free ribosomes are released into the cytosol and used within the cell. Since the cytosol contains high concentrations of glutathione and is, therefore, a reducing environment, proteins containing disulfide bonds, which are formed from oxidized cysteine residues, cannot be produced within it.

Membrane-bound ribosomes

When a ribosome begins to synthesize proteins needed in certain organelles, the ribosome making this protein can become "membrane-bound". In eukaryotic cells this happens in a region of the endoplasmic reticulum (ER) called the "rough ER". The newly produced polypeptide chains are inserted directly into the ER by the ribosome undertaking vectorial synthesis and are then transported to their destinations, through the secretory pathway. Bound ribosomes usually produce proteins that are used within the plasma membrane or are expelled from the cell via exocytosis.

Biogenesis

Main article: Ribosome biogenesis

In bacterial cells, ribosomes are synthesized in the cytoplasm through the transcription of multiple ribosome gene operons. In eukaryotes, the process takes place both in the cell cytoplasm and in the nucleolus, which is a region within the cell nucleus. The assembly process involves the coordinated function of over 200 proteins in the synthesis and processing of the four rRNAs, as well as assembly of those rRNAs with the ribosomal proteins.

Origin

The ribosome may have first originated as a protoribosome, possibly containing a peptidyl transferase centre (PTC), in an RNA world, appearing as a self-replicating complex that only later evolved the ability to synthesize proteins when amino acids began to appear. Studies suggest that ancient ribosomes constructed solely of rRNA could have developed the ability to synthesize peptide bonds. In addition, evidence strongly points to ancient ribosomes as self-replicating complexes, where the rRNA in the ribosomes had informational, structural, and catalytic purposes because it could have coded for tRNAs and proteins needed for ribosomal self-replication. Hypothetical cellular organisms with self-replicating RNA but without DNA are called ribocytes (or ribocells).

As amino acids gradually appeared in the RNA world under prebiotic conditions, their interactions with catalytic RNA would increase both the range and efficiency of function of catalytic RNA molecules. Thus, the driving force for the evolution of the ribosome from an ancient self-replicating machine into its current form as a translational machine may have been the selective pressure to incorporate proteins into the ribosome's self-replicating mechanisms, so as to increase its capacity for self-replication.

Heterogeneous ribosomes

Ribosomes are compositionally heterogeneous between species and even within the same cell, as evidenced by the existence of cytoplasmic and mitochondria ribosomes within the same eukaryotic cells. Certain researchers have suggested that heterogeneity in the composition of ribosomal proteins in mammals is important for gene regulation, i.e., the specialized ribosome hypothesis. However, this hypothesis is controversial and the topic of ongoing research.

Heterogeneity in ribosome composition was first proposed to be involved in translational control of protein synthesis by Vince Mauro and Gerald Edelman. They proposed the ribosome filter hypothesis to explain the regulatory functions of ribosomes. Evidence has suggested that specialized ribosomes specific to different cell populations may affect how genes are translated. Some ribosomal proteins exchange from the assembled complex with cytosolic copies suggesting that the structure of the in vivo ribosome can be modified without synthesizing an entire new ribosome.

Certain ribosomal proteins are absolutely critical for cellular life while others are not. In budding yeast, 14/78 ribosomal proteins are non-essential for growth, while in humans this depends on the cell of study. Other forms of heterogeneity include post-translational modifications to ribosomal proteins such as acetylation, methylation, and phosphorylation. Arabidopsis,Viral internal ribosome entry sites (IRESs) may mediate translations by compositionally distinct ribosomes. For example, 40S ribosomal units without eS25 in yeast and mammalian cells are unable to recruit the CrPV IGR IRES.

Heterogeneity of ribosomal RNA modifications plays a significant role in structural maintenance and/or function and most mRNA modifications are found in highly conserved regions. The most common rRNA modifications are pseudouridylation and 2'-O-methylation of ribose.

ncRNA therapy

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/NcRNA_therapy 

A majority of the human genome is made up of non-protein coding DNA. It infers that such sequences are not commonly employed to encode for a protein. However, even though these regions do not code for protein, they have other functions and carry necessary regulatory information.They can be classified based on the size of the ncRNA. Small noncoding RNA is usually categorized as being under 200 bp in length, whereas long noncoding RNA is greater than 200bp. In addition, they can be categorized by their function within the cell; Infrastructural and Regulatory ncRNAs. Infrastructural ncRNAs seem to have a housekeeping role in translation and splicing and include species such as rRNA, tRNA, snRNA.Regulatory ncRNAs are involved in the modification of other RNAs.

Timeline of ncRNA therapeutics

RNA Classification

Long non-coding RNA

Long non-coding RNA (LncRNA) are a type of RNA which is usually defined as transcripts which are greater than 200 base-pairs in length and not translated into proteins. This limitation distinguishes lncRNA from small non-coding RNAs which encompasses microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs. Long non-coding RNAs include lincRNAs, intronic ncRNAs, circular and linear ncRNA.

Long intergenic Non-coding RNA

Long intergenic Non-coding RNA (LincRNA) is defined as RNA transcripts that are longer than 200 nucleotides. These RNAs must not have open reading frames that encode proteins. The term “intergenic” refers to the identification of these transcripts from regions of the genome that do not contain protein-encoding genes. LncRNAs also contain promoter - or enhancer-associated RNAs that are gene proximal and can be either in the sense or antisense orientation.

Circular RNA

Circular RNA (CircRNA) are a novel class of endogenous noncoding RNAs and are characterized by their covalently closed loop structures. This class of ncRNA does not have a 5’ cap or 3’ Poly A tail. It has been hypothesized that cirRNAs may function as potential molecular markers for disease diagnosis and treatment and play an important role in the initiation and progression of human diseases.

Small non-coding RNA

Small non-coding RNA (sncRNA) are a type of RNA. which is usually defined as transcripts which are lesser than 200 base-pairs in length and not translated into proteins. This limitation distinguishes sncRNA from lncRNA. This class includes but is not limited to microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs.

microRNA

microRNA (miRNA) plays an important role in regulating gene expression. Majority of miRNAs are transcribed from DNA sequences into primary miRNAs. These primary miRNAs are further processed into precursor miRNAs, and finally into mature miRNAs. The miRNAs in most cases interact with the 3’ UTR region of target to induce mRNA degradation and translational repression. Interactions of miRNAs with other regions, including the 5’ UTR, coding sequence, and gene promoters have also been reported. Under certain conditions, miRNAs are also able to activate translation or regulate transcription, but this is dependent on factors such as location of the effect. This process of interaction is very dynamic and dependent on multiple factors.

Ribosomal RNA

Ribosomal RNA (rRNA) includes non-coding RNAs that play essential roles in rRNA regulation. Ribosomal RNA (rRNA) takes part in protein synthesis. Occasional RNA molecules act catalytically, as RNA enzymes (ribozymes) or take part in protein export. The most important ribozyme is the major rRNA of the large subunit of the ribosome (28s rRNA in eukaryotes). It is now accepted that 28S rRNA catalyzes the critical step in polypeptide synthesis in addition to playing a major structural role.

Small nuclear RNA and small nucleolar RNA

Small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) are widely known to guide the nucleotide modifications and processing of rRNA. Both snRNA and snoRNA are categorized into a class of small RNA molecules that are present in the nucleus. However, they vary a lot by function. snRNA are 80-350nucletides long while snoRNA are 80-1000 nucleotides long in yeast. snRNA plays a critical role in regulating the pre-mRNA silencing. On the other hand, snoRNAs are involved in mRNA editing, modification of the rRNA and tRNA, and genome imprinting. Major function of snoRNA includes the maturation of rRNA during ribosomal formation. Small nuclear and small nucleolar RNAs are critical components of snRNPs and snoRNPs and play an essential role in the maturation of, respectively, mRNAs and rRNAs within the nucleus of eukaryotic cells. Both snRNA and snoRNA are involved in modifying RNA just after transcription. snRNA can be found in splicing speckles and Cajal bodies of the nucleus of the cell.snRNA and snoRNA requires a phosphorylated adaptor for nuclear export (PHAX) to get transported to the site of action within the nucleus.

Transfer RNA

Transfer RNA (tRNA) helps decode a messenger RNA sequence into a protein. They function at specific sites within the ribosome during translation (the process going from code to protein). Within the mRNA molecule we have three nucleotides in length codons. These codons all have a unique universal code which represents a particular amino acid. tRNAs can be classified as an adaptor molecule, being typically 76 to 90 nucleotides in length.

History

Non-coding RNA

DNA purification in 1869 by Dr. Friedrich Miescher’s, from salmon sperm and pus cells guided the scientists towards the presence of additional molecules in the cell except for proteins. Miescher identified the presence of a highly acidic molecule that he isolated from the pus cells and labeled it “nuclein”. The term was coined as the DNA isolated by Miescher was not protein and was derived from the nucleus of the cell. It wasn’t until 1944, when Oswald Avery proposed the DNA as a genetic carrier of information that the Miescher discovery was brought back to light.

Following the X-ray crystallography, by Rosalind Franklin and the determination of DNA double helix by Watson and Crick in 1953, further enhanced the understanding of DNA structure and allowed for the establishment of central dogma of molecular biology. However, one of the flaws with central dogma was the postulation that information flow proceeds from DNA to RNA to protein, which hinders the understanding of different regulatory mechanisms.

In 1955, George Palade identified the first ncRNA as a part of the large ribonucleoprotein complex (RNP). The second class of ncRNA to be discovered was transfer RNA (tRNA) in 1957. However, the first regulatory ncRNA was a microRNA discovered in 1988 from E.coli and was labeled as micF. On other hand, the first eukaryotic microRNA was discovered in C.elegans in 1993. It was derived from gene lin-4 and was identified as a small RNA molecule (as compared to longer mRNA molecules) forming stem-loop structures. This structure gets further modified to generate a shorter RNA that is complementary to the 3’UTR region of lin-14 transcript. This pathway allowed for a better understanding of different post translational gene silencing pathways. Since then, many other miRNAs have been discovered.

Detailed understanding of the mechanism behind this post translational silencing pathway was established in 2001 by Thomas Tuschl. It was discovered that the double stranded RNA gets processed into a shorter 25 nucleotides long fragment which is then modified into a short hairpin like structure by Drosha complex. The molecule is then diced by dicer enzymes into a functional double stranded RNA (dsRNA). These are then loaded onto the RISC complex which then finds and cleaves the targeted mRNA of interest in the cytoplasm.

It wasn’t until 1989 that the imprinting genes were discovered and the genome imprinting was established. The first two genomic imprinting genes were paternally expressed Igf2r and H19. These were both discovered independently in mice and were localized to chromosome 7. H19 is peculiar as it functions as a lncRNA but undergoes modifications similar to that of pre-mRNA processing such as splicing, 3’ polyadenylation and is transcribed by RNA polymerase II. This lncRNA plays a significant role in mice embryonic development and can be lethal if expressed during prenatal stages. More lncRNAs have been discovered in eukaryotes overtime. One such discovery that allowed for better understanding between H19 functions was a lncRNA called XIST (X inactive-specific transcript).

ncRNA drugs and therapy

The first ncRNA therapeutic drug approved by food and drug administration (FDA) (1998) and the European medicine agency (EMA) (1999) is called Fomivirsen or Vitravene. The target organ is the eye and works against the cytomegalovirus (CMV) retinitis in immunocompromised patients. The drug functions as an antisense oligonucleotide and binds to the complementary sequence of the mRNA that inhibits the replication of human cytomegalovirus. This therapy can also be categorized as Antisense oligonucleotide (ASO) therapy. There have been many ASO RNA therapeutics that have been approved by FDA and/or EMA over the years, but it wasn’t until 2018 that the EMA approved the drug called Patisiran/Onpattro. The drug uses ds-siRNA as a mechanism of action and is deemed effective against hereditary transthyretin amyloidosis. The mechanism specifically targets the Transthyretin (TTR) mRNA. RNA therapeutic targets are not limited to mature mRNA but have been used to target mRNA at different stages of maturation. One such example is Nusinersen (Spinaraza), it functions as an ASO and targets pre-mRNA before splicing that corresponds to Survival of motor neuron 2 gene (SMN 2). This drug therapy was approved by FDA and EMA in 2016 and 2017 respectively. There are some drugs that have been approved by FDA and not by EMA. This can be seen in the case of an ASO type therapeutics called Eteplirsen (Exondys51) which has been approved by FDA in 2016 but not by EMA. It targets pre-mRNA corresponding to Dystrophin (DMD) and works against Duchenne muscular dystrophy. There are many additional therapeutics that have been developed and are either in phase I or II of the clinical trials. Current RNA therapeutics in clinical trials range from a variety of target organs and diseases ranging from skin (potential treatment for disease such as keloid) to tumors (squamous cell lung cancer).

To date, for both the FDA and the EMA, ncRNAs are considered as "simple" medical products because of their production by chemical synthesis. When some of them, produced biologically (known as bioengineered ncRNA agents: BERAs), will be put on the market, the status of biological medical products will be applied, which could lead to inconsistencies in the legislation.

Applications

Antisense oligonucleotides

Antisense Oligonucleotide Use in ncRNA therapy

Antisense oligonucleotides (ASOs) are single-stranded DNA molecules with full complementarity to one select target mRNA and may act by blocking protein translation (via steric hindrance), causing mRNA degradation (via RNase H-cleavage) or changing pre-mRNA splicing. These short oligonucleotides have already been approved by the FDA for ten genetic disorders and many are currently in the pipeline to be approved/tested. Using oligonucleotide technology, we are now able to control protein expression via RNA interference, and are able to affect previously defined “undruggable” proteins. Even though this therapy has a lot of promise and potential, it comes with many limitations.

Compared to siRNA and microRNA, ASOs are more versatile in reducing protein expression, they have the ability to also enhance target translation. ASOs can also be customized with ease and accuracy, allowing for the targeting of virtually any mutated gene. This allows for a greater level of application in the field of precision and personalized medicine. The main challenge of ASO therapies to specific tissues and cellular uptake is what poses a great challenge and limitation. Liposomal delivery is one such way to overcome such issues. Liposomal delivery system comes with its own share of limitations. Serum proteins in the bloodstream destabilize the lipoprotein. This destabilization leads to the depletion of protein and exposing cargo to the unstable environment. This hindrance can be overcome by using PEGs (poly(ethylene glycol) . However, PEGs are not biodegradable causing them to accumulate within the body leading to adverse effects and causing hypersensitivity. In addition, multiple rounds of therapy with PEGs can lead to the formation of PEG antibodies, which can lead to lack of efficiency in preventing the rupture of the liposome that it is attached to. Using immunoliposomes it has been shown that targeting can be more specific as by using antibody’s specific to the protein of expression in that area, it results in the ASO drug directly impact the target site and nowhere else. Moreover, immunoliposomes are slow to dissociate leading to precise release of the ASO drug which they encapsulate.

LncRNA as a therapeutic approach

Long noncoding RNAs (lncRNAs) are large transcripts (more than 200 nucleotides long) that have similar mechanism of synthesis as that of mRNAs but unlike mRNAs, lncRNAs are not translated to a protein. lncRNA contains interactor elements and structural elements. Interactor elements directly interact with other nucleic acids or proteins while the structural elements indicate the ability of some lncRNAs to form secondary and/or tertiary structures. This ability of the lncRNAs to interact with nucleic acids using its interactor elements and its ability to interact with protein using its secondary structures allows it to function in a more diverse manner than other ncRNAs such as miRNA (microRNA). LncRNA has been established to play a role in gene regulation by influencing the ability of specific regions of the gene to bind to transcriptional elements and different epigenetic modifications. One such example can be seen in the case X inactive specific transcript (XIST). In humans, 46,XX females carry an extra X chromosome (155Mb of DNA) compared to 46,XY males. To overcome this dosage imbalance, one X chromosome is randomly inactivated in human females at around the 2-8 cell stage of embryo development. This inactivation is very stable across cell divisions due to epigenetic contributions both during the initial silencing and the subsequent maintenance of the inactive X chromosome (Xi). This inactivation is carried by the lncRNA, XIST. XIST is produced in cis and inactivates the X-chromosome that it has been generated from. The inactive X chromosome can be observed as a condensed heterochromatin structure called “Barr Body”. A study in 2013 utilized this ability of XIST as a potential therapeutic approach for treatment of trisomy 21. Trisomy 21 is commonly known as down syndrome and is caused due to presence of an additional copy of chromosome 21. The study was one of its kind as no other studies have been able to incorporate the XIST gene into a chromosome due to its large size. The study incorporated the XIST into one of the chromosomes 21 in the cells gathered from patients with down syndrome. The study was able to observe the inactivation of one of chromosome 21 in the form of a condensed heterochromatin and labeled it as a chromosome 21 barr body. Such experiments have shown to work in cells in the lab setting although no lncRNA based therapeutics are in clinical trials. The implications of such work can bring trisomy 21 and other chromosomal disorders in the realm of consideration for future gene therapy research.

Challenges

One of the major issues that hinders the ncRNA therapy is the stability of the single stranded RNA molecule. RNA is typically single stranded therefore slightly unstable as compared to dsDNA molecules. This however can be overcome by fabricating the single stranded RNA to double stranded RNA(dsRNA). This is quite effective as the dsRNA is more stable at room temperature and has a longer shelf life. Second major issue is the cell/tissue/organ specific targeting of the RNA molecules. Generally, this is overcome by containing the dsRNA in a lipid nanoparticle and using that as a ligand to bind to a receptor on the surface of the target cell. The lipid particles are taken into the liver cells through their specific receptors and this mechanism seems to be effective at targeting the liver cells/cancer. Another organ with a relatively easy delivery mechanism is the eye. This requires an invasive technique of directly injecting the ncRNA of interest directly into the eye. These techniques are not only invasive but also don’t ensure if all the cells in the target organ are being targeted by the ncRNA of interest. Additional issues arise once the RNA molecule enters the cell. One of the issues being the immune system. Our immune system can recognize RNA using the intracellular pathogen associated molecular pattern (PAMP) receptors and extracellular toll-like receptors (TLR). Activation of the receptors leads to a cytokine (IFNy-Interferon gamma) mediated immune response. Common applications to overcome the immune response include second generation chemical modifications. This process includes the introduction of small one at a time chemical modifications to avoid the immune response. However, there are some reports of adverse immune responses in clinical trials employing such modified reagents. There’s no fixed answer to issues with immunogenicity and ncRNA therapy. Modified adenovirus vectors have been used extensively in many clinical trials as a ncRNA delivery mechanism. In particular, adenovirus vector is considered an efficient delivery system due to its stability within live cells and non-pathogenicity. Even though viral transfections have achieved significant results in basic research, one of the issues is the non-specificity leading to off target transfections. Further research needs to be done to improve the accuracy of viral transfections for future tests and clinical trials.

ASO Guidelines

In December 2021, the FDA came up with a draft guidance for the use of ASO drug products. This draft guidance was directed towards sponsor-investigators who are developing individualized investigational antisense oligonucleotides (ASO) drug products for severely debilitating or life threatening diseases. Severely debilitating corresponds to a disease or condition that causes major irreversible morbidity. However, life-threatening is defined as the disease or condition has a likelihood of death unless the course of treatment leads to an endpoint of survival. Usually individuals that have a severely debilitating life threatening disease don't have any alternative treatment options, and their diseases will be rapidly progressing, leading to an early death and/or devastating or irreversible morbidity within a short time frame without treatment.

Drug development is usually targeted for a large number of individuals, in this case that is not possible because of the specificity of the mechanism of action of the ASO combined with the rarity of the treatment-amenable patient population. Under FDA regulations, a protocol under which an individual ASO product is administered to a human subject must be reviewed and approved by an institutional review board (IRB) before it can be administered to human subjects. When the individual is a child, additional safeguards need to be identified in order to prevent any developmental issues from occurring that may affect the life of the individual. The sponsor-investigator needs to get informed consent from the individual or from the person who is responsible for the individual. The consent needs to include a description of reasonably foreseeable risks or discomforts as part of the use of the ASO drug. The sponsor also needs to get individuals clinical and genetic diagnosis to confirm that the ASO will be beneficial. The analysis may be through gene sequencing, enzymatic analysis, biochemical testing, imaging evaluations. All results need to be included in the application. Also the sponsor needs to include evidence that establishes the role of the gene variant targeted by the ASO drug. The sponsor/investigator need to also provide evidence that the identified gene variant or variants are unique to the individual.

The guidance suggests that the starting dose should be based on available non-clinical data that has been collected from model organisms or in vitro studies and should be in correlation with other ASO drug product dosing information that is available. At the starting dose, pharmacological effects are expected. Furthermore, It is advised that a dosing escalation method be utilized. This includes the step of escalating the dodge from its initial dose based on pharmacodynamic effects and/or trial participants' response to the ASO.

In addition, protocols submitted to the FDA need to have a clear dosing plan and justification for selecting the starting dose, dosing interval, and plan for dose escalation or dose reduction based on clinical pharmacodynamic effects of the drug on the individual. Also all anticipated outcomes should be included in the drug plan when submitted to the FDA. It is extremely important for the investigators to monitor the patient closely during dose escalation. During the escalation period, adequate time should be provided in order to see therapeutic results. It is advised that the investigator not make concurrent changes to the dosing interval along with the dose without justification. The submitted plan should include a de-escalation/discontinuation plan if toxicity is observed. All drug administration needs to take place in an inpatient setting just to get a grasp of the adverse effects the drug may have. Once drug toxicity, beneficiancy and adverse effects are identified, the drug can be administered in an outpatient manner as long as the same concentration of drug is administered.

RNA therapeutics

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/RNA_therapeutics 

 

See also: DNA vaccine and RNA vaccine

RNA therapeutics are a new class of medications based on ribonucleic acid (RNA). Research has been working on clinical use since the 1990s, with significant success in cancer therapy in the early 2010s. In 2020 and 2021, mRNA vaccines have been developed globally for use in combating the coronavirus disease (COVID-19 pandemic). The Pfizer–BioNTech COVID-19 vaccine was the first mRNA vaccine approved by a medicines regulator, followed by the Moderna COVID-19 vaccine, and others.

The main types of RNA therapeutics are those based on messenger RNA (mRNA), antisense RNA (asRNA), RNA interference (RNAi), RNA activation (RNAa) and RNA aptamers. Of the four types, mRNA-based therapy is the only type which is based on triggering synthesis of proteins within cells, making it particularly useful in vaccine development. Antisense RNA is complementary to coding mRNA and is used to trigger mRNA inactivation to prevent the mRNA from being used in protein translation. RNAi-based systems use a similar mechanism, and involve the use of both small interfering RNA (siRNA) and micro RNA (miRNA) to prevent mRNA translation and/or degrade mRNA. Small activating RNA (saRNA) represents a novel class of RNA therapeutics that upregulates gene expression via the RNAa mechanism, offering a unique mechanism compared to other RNA-based therapies. However, RNA aptamers are short, single stranded RNA molecules produced by directed evolution to bind to a variety of biomolecular targets with high affinity thereby affecting their normal in vivo activity.

RNA is synthesized from template DNA by RNA polymerase with messenger RNA (mRNA) serving as the intermediary biomolecule between DNA expression and protein translation. Because of its unique properties (such as its typically single-stranded nature and its 2' OH group) and its ability to adopt many different secondary/tertiary structures, both coding and noncoding RNAs have attracted attention in medicine. Research has begun to explore RNAs potential to be used for therapeutic benefit, and unique challenges have occurred during drug discovery and implementation of RNA therapeutics.

mRNA

Messenger RNA (mRNA) is a single-stranded RNA molecule that is complementary to one of the DNA strands of a gene. An mRNA molecule transfers a portion of the DNA code to other parts of the cell for making proteins. DNA therapeutics needs access to the nucleus to be transcribed into RNA, and its functionality depends on nuclear envelope breakdown during cell division. However, mRNA therapeutics do not need to enter into the nucleus to be functional since it will be translated immediately once it has reached to the cytoplasm. Moreover, unlike plasmids and viral vectors, mRNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis, making them suitable for use in cancer vaccines, tumor immunotherapy and infectious disease prevention.

Discovery and development

In 1953, Alfred Day Hershey reported that soon after infection with phage, bacteria produced a form of RNA at a high level and this RNA was also broken down rapidly. However, the first clear indication of mRNA was from the work of Elliot Volkin and Lazarus Astrachan in 1956 by infecting E.coli with T2 bacteriophages and putting them into the medium with ³²P. They found out that the protein synthesis of E.coli was stopped and phage proteins were synthesized. Then, in May 1961, their collaborated researchers Sydney Brenner, François Jacob, and Jim Watson announced the isolation of mRNA. For a few decades after mRNA discovery, people focused on understanding the structural, functional, and metabolism pathway aspects of mRNAs. However, in 1990, Jon A. Wolff demonstrated the idea of nucleic acid-encoded drugs by direct injecting in vitro transcribed (IVT) mRNA or plasmid DNA (pDNA) into the skeletal muscle of mice which expressed the encoded protein in the injected muscle.

Once IVT mRNA has reached the cytoplasm, the mRNA is translated instantly. Thus, it does not need to enter the nucleus to be functional. Also, it does not integrate into the genome and therefore does not have the risk of insertional mutagenesis. Moreover, IVT mRNA is only transiently active and is completely degraded via physiological metabolic pathways. Due to these reasons, IVT mRNA has undergone extensive preclinical investigation.

Mechanisms

In vitro transcription (IVT) is performed on a linearized DNA plasmid template containing the targeted coding sequence. Then, naked mRNA or mRNA complexed in a nanoparticle will be delivered systemically or locally. Subsequently, a part of the exogenous naked mRNA or complexed mRNA will go through cell-specific mechanisms. Once in the cytoplasm, the IVT mRNA is translated by the protein synthesis machinery.

There are two identified RNA sensors, toll-like receptors (TLRs) and the RIG-I-like receptor family. TLRs are localized in the endosomal compartment of cells, such as DCs and macrophages. RIG-I-like family is as a pattern recognition receptor (PRR). However, the immune response mechanisms and process of mRNA vaccine recognition by cellular sensors and the mechanism of sensor activation are still unclear.

Applications

Cancer immunotherapy

In 1995, Robert Conry demonstrated that intramuscular injection of naked RNA encoding carcinoembryonic antigen elicited antigen-specific antibody responses. Then, it was elaborated by demonstrating that dendritic cells(DCs) exposed to mRNA coding for specific antigens or to total mRNA extracted from tumor cells and injected into tumor-bearing mice induced T cell immune responses and inhibited the growth of tumors. Then, researchers started to approach mRNA transfected DCs using vaccines based on ex vivo IVT mRNA-transfected DCs. Meanwhile, Argos Therapeutics had initiated a Phase III clinical trial using DCs with advanced renal cell carcinoma in 2015 (NCT01582672) but it was terminated due to the lack of efficacy.

For further application, IVT mRNA was optimized for in situ transfections of DCs in vivo. It improved the translation efficiency and stability of IVT mRNA and enhanced the presentation of the mRNA-encoded antigen on MHC class I and II molecules. Then, they found out that the direct injection of naked IVT mRNA into lymph nodes was the most effective way to induce T cell responses. Based on this discovery, first-in-human testing of the injection of naked IVT mRNA encoding cancer antigens by BioNTech has started with patients with melanoma (NCT01684241).

Recently, the new cancer immunotherapy, the combining of self-delivering RNA(sd-rxRNA) and adoptive cell transfer(ACT) therapy, was invented by RXi Pharmaceuticals and the Karolinska Institute. In this therapy, the sd-rxRNA eliminated the expression of immunosuppressive receptors and proteins in therapeutic immune cells so it improved the ability of immune cells to destroy the tumor cells. Then, the PD-1 targeted sd-rxRNA helped increasing the anti-tumor activity of tumor-infiltrating lymphocytes (TIL) against melanoma cells. Based on this idea, the mRNA-4157 has been tested and passed phase I clinical trial.

Cytosolic nucleic acid-sensing pathways can enhance immune response to cancer. RIG-I agonist, stem loop RNA (SLR) 14. Tumor growth was significantly delayed and extended survival in mice. SLR14 improved antitumor efficacy of anti-PD1 antibody over single-agent treatment. SLR14 was absorbed by CD11b+ myeloid cells in the tumor microenvironment. Genes associated with immune defense were significantly up-regulated, along with increased CD8+ T lymphocytes, NK cells, and CD11b+ cells. SLR14 inhibited nonimmunogenic B16 tumor growth, leaving immune memory.

Vaccines

Main article: RNA vaccine

In 1993, the first success of an mRNA vaccine was reported in mice, by using liposome-encapsulated IVT mRNA which is encoding the nucleoprotein of influenza that induced virus-specific T cells. Then, IVT mRNA was formulated with synthetic lipid nanoparticles and it induced protective antibody responses against the respiratory syncytial virus(RSV) and influenza virus in mice.

There are a few different types of IVT mRNA-based vaccine development for infectious diseases. One of the successful types is using self-amplifying IVT mRNA that has sequences of positive-stranded RNA viruses. It was originally developed for a flavivirus and it was workable with intradermal injection. One of the other ways is injecting a two-component vaccine which is containing an mRNA adjuvant and naked IVT mRNA encoding influenza hemagglutinin antigen only or in combination with neuraminidase encoding IVT mRNA.

For example, for the HIV treatment, vaccines are using DCs transfected with IVT mRNA that is encoding HIV proteins. There are a few phase I and II clinical trials using IVT mRNA encoding combinations and it shows that antigen-specific CD8+ and CD4+ T cell responses can be induced. However, no antiviral effects have been observed in the clinical trial.

One of the other mRNA vaccines is for COVID-19. The Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) outbreaks in December 2019 and spread all over the world, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). The Moderna COVID-19 vaccine, manufactured by Moderna since 2020, is a lipid nanoparticle (LNP) encapsulated mRNA-based vaccine that encodes for a full-length, prefusion stabilized spike(S)-2P antigen of SARS-CoV-2 with a transmembrane anchor.

Anti-viral

In 2021, SLR14 was reported to prevent infection in the lower respiratory tract and severe disease in an interferon type I (IFN-I)–dependent manner in mice. Immunodeficient mice with chronic SARS-CoV-2 infection experienced near-sterilizing innate immunity with no help from the adaptive immune system.

Tissue regeneration

A 2022 study by researchers from the Mayo Clinic, Maastricht University, and Ethris GmBH, a biotech company that focuses on RNA therapeutics, found that chemically modified mRNA encoding BMP-2 promoted dosage-dependent healing of femoral osteotomies in male rats. The mRNA molecules were complexed within nonviral lipid particles, loaded onto sponges, and surgically implanted into the bone defects. They remained localized around the site of application. Compared to receiving rhBMP-2 directly, bony tissues regenerated after mRNA treatment displayed superior strength and less formation of massive callus.

Limitations

There are many challenges for the successful translation of mRNA into drugs because mRNA is a very large and heavy molecule(10^5 ~ 10^6 Da). Moreover, mRNA is unstable and easily degraded by nucleases, and it also activates the immune systems. Furthermore, mRNA has a high negative charge density and it reduces the permeation of mRNA across cellular membranes. Due to these reasons, without the appropriate delivery system, mRNA is degraded easily and the half-life of mRNA without a delivery system is only around 7 hours. Even though some degrees of challenges could be overcome by chemical modifications, delivery of mRNA remains an obstacle. The methods that have been researched to improve the delivery system of mRNA are using microinjection, RNA patches (mRNA loaded in a dissolving micro-needle), gene gun, protamine condensation, RNA adjuvants, and encapsulating mRNA in nanoparticles with lipids.

Even though In Vitro Translated (IVT) mRNA with delivery agents showed improved resistance against degradation, it needs more studies on how to improve the efficiency of the delivery of naked mRNA in vivo.

Approved RNA Therapeutics

• patisiran
• givosiran
• lumasiran
• inclisiran

Antisense RNA

Antisense RNA is the non-coding and single-stranded RNA that is complementary to a coding sequence of mRNA. It inhibits the ability of mRNA to be translated into proteins. Short antisense RNA transcripts are produced within the nucleus by the action of the enzyme Dicer, which cleaves double-stranded RNA precursors into 21–26 nucleotide long RNA species.

There is an antisense-based discovery strategy, rationale and design of screening assays, and the application of such assays for screening of natural product extracts and the discovery of fatty acid condensing enzyme inhibitors. Antisense RNA is used for treating cancer and inhibition of metastasis and vectors for antisense sequestration. Particularly MicroRNAs(miRs) 15 and 16 to a patient in need of the treatment for diagnosis and prophylaxis of cancer. Antisense drugs are based on the fact that antisense RNA hybridizes with and inactivates mRNA. These drugs are short sequences of RNA that attach to mRNA and stop a particular gene from producing the protein for which it encodes. Antisense drugs are being developed to treat lung cancer, diabetes and diseases such as arthritis and asthma with a major inflammatory component. It shows that the decreased expression of MLLT4 antisense RNA 1 (MLLT4‑AS1) is a potential biomarker and a predictor of a poor prognosis for gastric cancer. So far, applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms have been explored.

Non-viral vectors, virus vectors and liposomes have been used to deliver the antisense RNA through the cell membrane into the cytoplasm and nucleus. It has been found that the viral vector based delivery is the most advantageous among different delivery systems because it has a high transfection efficacy. However, it is difficult to deliver antisense RNA only to the targeted sites. Also, due to the size and the stability issues of antisense RNA, there are some limitations to its use. To improve the delivery issues, chemical modifications, and new oligonucleotide designs have been studied to enhance the drug distribution, side effects, and tolerability.

RNAi

Interfering RNA are a class of short, noncoding RNA that act to translationally or post-translationally repress gene expression. Their discovery and subsequent identification as key effectors of post-transcriptional gene regulation have made small interfering RNA (siRNA) and micro RNA (miRNA) potential therapeutics for systemic diseases. The RNAi system was originally discovered in 1990 by Jorgensen et al., who were doing research involving the introduction of coloration genes into petunias, and it is thought that this system originally developed as a means of innate immunity against double-stranded RNA viruses.

siRNA

A schematic of the mechanism for siRNA and miRNA gene regulation in vivo.

Small interfering (siRNA) are short, 19-23 base-pair (with a 3' overhang of two nucleotides), double-stranded pieces of RNA that participate in the RNA-induced silencing complex (RISC) for gene silencing. Specifically, siRNA is bound by the RISC complex where it is unwound using ATP hydrolysis. It is then used as a guide by the enzyme "Slicer" to target mRNAs for degradation based on complementary base-pairing to the target mRNA. As a therapeutic, siRNA is able to be delivered locally, through the eye or nose, to treat various diseases. Local delivery benefits from simple formulation and drug delivery and high bioavailability of the drug. Systemic delivery is necessary to target cancers and other diseases. Targeting the siRNA when delivered locally is one of the main challenges in siRNA therapeutics. While it is possible to use intravenous injection to deliver siRNA therapies, concerns have been raised about the large volumes used in the injection, as these must often be ~20-30% of the total blood volume. Other methods of delivery include liposome packaging, conjugation to membrane-permeable peptides, and direct tissue/organ electroporation. Additionally, it has been found that exogeneous siRNAs only last a few days (a few weeks at most in non-dividing cells) in vivo. If siRNA is able to successfully reach its target, it has the potential to therapeutically regulate gene expression through its ability to base-pair to mRNA targets and promote their degradation through the RISC system. Currently, siRNA-based therapy is in a phase I clinical trial for the treatment of age-related macular degeneration, although it is also being explored for use in cancer therapy. For instance, siRNA can be used to target mRNAs that code for proteins that promote tumor growth such as the VEGF receptor and telomerase enzyme.

miRNA

Micro RNAs (miRNAs) are short, ~19-23 base pair long RNA oligonucleotides that are involved in the microRNA-induced silencing complex. Specifically, once loaded onto the ARGONAUTE enzyme, miRNAs work with mRNAs to repress translation and post-translationally destabilize mRNA. While they are functionally similar to siRNAs, miRNAs do not require extensive base-pairing for mRNA silencing (can require as few as seven base-pairs with target), thus allowing them to broadly affect a wider range of mRNA targets. In the cell, miRNA uses switch, tuning, and neutral interactions to finely regulate gene repression. As a therapeutic, miRNA has the potential to affect biochemical pathways throughout the organism.

With more than 400 miRNA identified in humans, discerning their target gene for repression is the first challenge. Multiple databases have been built, for example TargetScan, using miRNA seed matching. In vitro assays assist in determining the phenotypic effects of miRNAs, but due to the complex nature of gene regulation not all identified miRNAs have the expected effect. Additionally, several miRNAs have been found to act as either tumor suppressors or oncogenes in vivo, such as the oncogenic miR-155 and miR-17-92.

In clinical trials, miRNA are commonly used as biomarkers for a variety of diseases, potentially providing earlier diagnosis as well as disease progression, stage, and genetic links. Phase 1 and 2 trials currently test miRNA mimics (to express genes) and miRNA (to repress genes) in patients with cancers and other diseases. In particular, mimic miRNAs are used to introduce miRNAs that act as tumor suppressors into cancerous tissues, while miRNA antagonists are used to target oncogenic miRNAs to prevent their cancer-promoting activity. Therapeutic miRNA is also used in addition to common therapies (such as cancer therapies) that are known to overexpress or destabilize the patient miRNA levels. An example of one mimic miRNA therapy that demonstrated efficacy in impeding lung cancer tumor growth in mouse studies is miR-34a.

One concerning aspect of miRNA-based therapies is the potential for the exogeneous miRNA to affect miRNA silencing mechanisms within normal body cells, thereby affecting normal cellular biochemical pathways. However, in vivo studies have indicated that miRNAs display little to no effect in non-target tissues/organs.

Small activating RNAs (saRNAs)

Main article: Small activating RNA

Small activating RNAs (saRNAs) are short double-stranded RNA molecules (typically 19–21 nucleotides in length) that induce transcriptional activation of target genes through a process known as RNA activation (RNAa). Unlike RNA interference (RNAi), which silences gene expression, saRNAs upregulate gene expression by targeting promoter regions of DNA and recruiting transcriptional machinery.

The mechanism of RNAa involves the formation of an RNA-induced transcriptional activation (RITA) complex. This complex includes Argonaute proteins (particularly Ago2), RNA helicase A (RHA), and other transcriptional coactivators, which facilitate the activation of RNA polymerase II at the targeted promoter. This process is often associated with epigenetic changes, such as histone modifications, that promote active transcription.

saRNAs have demonstrated potential in preclinical studies for treating diseases caused by insufficient gene expression, such as cancer and metabolic disorders. For example, saRNAs have been used to reactivate tumor suppressor genes in cancer cells, offering a promising therapeutic approach. Additionally, saRNAs are being explored for their ability to upregulate genes involved in metabolic regulation, neurodegenerative diseases, and other conditions.

An example of an saRNA therapeutic in clinical development is MTL-CEBPA, which targets the CEBPA gene to treat liver cancer. This drug, developed by MiNA Therapeutics, has shown promise in early-phase clinical trials. Another saRNA therapeutic, RAG-01, developed by Ractigen Therapeutics, is being investigated for the treatment of non-muscle invasive bladder cancer (NMIBC) and has shown promising early complete responses (CRs) in Phase I trial for BCG-unresponsive patients.

saRNAs represent a significant advancement in RNA therapeutics, expanding the scope of RNA-based therapies to include gene activation in addition to gene silencing.

RNA aptamers

An RNA aptamer known as "Spinach" was created as a fluorescent imaging tool in vivo. Its fluorescent activity is based upon binding to 3,5-difluoro-4-hydroxybenzylidene imidazolidinone (DFHBI).

Broadly, aptamers are small molecules composed of either single-stranded DNA or RNA and are typically 20-100 nucleotides in length, or ~3-60 kDa. Because of their single-stranded nature, aptamers are capable of forming many secondary structures, including pseudoknots, stem loops, and bulges, through intra-strand base pairing interactions. The combinations of secondary structures present in an aptamer confer it a particular tertiary structure which in turn dictates the specific target the aptamer will selectively bind to. Because of the selective binding ability of aptamers, they are considered a promising biomolecule for use in pharmaceuticals. Additionally, aptamers exhibit tight binding to targets, with dissociation constants often in the pM to nM range. Besides their strong binding ability, aptamers are also valued because they can be used on targets that are not capable of being bound by small peptides generated by phage display or by antibodies, and they are able to differentiate between conformational isomers and amino acid substitutions. Also, because aptamers are nucleic-acid based, they can be directly synthesized, eliminating the need for cell-based expression and extraction as is the case in antibody production. RNA aptamers in particular are capable of producing a myriad of different structures, leading to speculations that they are more discriminating in their target affinity compared to DNA aptamers.

Discovery and development

Aptamers were originally discovered in 1990 when Lary Gold and Craig Tuerk utilized a method of directed evolution known as SELEX to isolate a small single stranded RNA molecule that was capable of binding to T4 bacteriophage DNA polymerase. Additionally, the term “aptamer” was coined by Andrew Ellington, who worked with Jack Szostak to select an RNA aptamer that was capable of tight binding to certain organic dye molecules. The term itself is a conglomeration of the Latin “aptus” or “to fit” and the Greek “meros” or “part."

RNA aptamers are not so much “created” as “selected.” To develop an RNA aptamer capable of selective binding to a molecular target, a method known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is used to isolate a unique RNA aptamer from a pool of ~10^13 to 10^16 different aptamers, otherwise known as a library. The library of potential aptamer oligonucleotides is then incubated with a non-target species so as to remove aptamers that exhibit non-specific binding. After subsequent removal of the non-specific aptamers, the remaining library members are then exposed to the desired target, which can be a protein, peptide, cell type, or even an organ (in the case of live animal-based SELEX). From there, the RNA aptamers which were bound to the target are transcribed to cDNA which then is amplified through PCR, and the PCR products are then re-transcribed to RNA. These new RNA transcripts are then used to repeat the selection cycle many times, thus eventually producing a homogeneous pool of RNA aptamers capable of highly specific, high-affinity target binding.

Examples

RNA aptamers can be designed to act as antagonists, agonists, or so-called ”RNA decoy aptamers." In the case of antagonists, the RNA aptamer is used either to prevent binding of a certain protein to its cell membrane receptor or to prevent the protein from performing its activity by binding to the protein's target. Currently, the only RNA aptamer-based therapies that have advanced to clinical trials act as antagonists. When RNA aptamers are designed to act as agonists, they promote immune cell activation as a co-stimulatory molecule, thus aiding in the mobilization of the body's own defense system. For RNA decoy aptamers, the synthetic RNA aptamer resembles a native RNA molecule. As such, proteins(s) which bind to the native RNA target instead bind to the RNA aptamer, possibly interfering with the biomolecular pathway of a particular disease. In addition to their utility as direct therapeutic agents, RNA aptamers are also being considered for other therapeutic roles. For instance, by conjugating the RNA aptamer to a drug compound, the RNA aptamer can act as a targeted delivery system for that drug. Such RNA aptamers are known as ApDCs. Additionally, through conjugation to radioisotope or a fluorescent dye molecule, RNA aptamers may be useful in diagnostic imaging.

Because of the SELEX process utilized to select RNA aptamers, RNA aptamers can be generated for many potential targets. By directly introducing the RNA aptamers to the target during SELEX, a very selective, high-affinity, homogeneous pool of RNA aptamers can be produced. As such, RNA aptamers can be made to target small peptides and proteins, as well as cell fragments, whole cells, and even specific tissues. Examples of RNA aptamer molecular targets and potential targets include vascular endothelial growth factor, osteoblasts, and C-X-C Chemokine Ligand 12 (CXCL2).

The chemical structure of the RNA aptamer known as Pegaptanib, a treatment for age-related macular degeneration.

An example of an RNA aptamer therapy includes Pegaptanib (aka Macugen ® ), the only FDA-approved RNA aptamer treatment. Originally approved in 2004 to treat age-related macular degeneration, Pegaptanib is a 28 nucleotide RNA aptamer that acts as a VEGF antagonist. However, it is not as effective as antibody-based treatments such as bevacizumab and ranibizumab. Another example of an RNA aptamer therapeutic is NOX-A12, a 45 nucleotide RNA aptamer that is in clinical trials for chronic lymphocytic leukemia, pancreatic cancer, as well as other cancers. NOX-A12 acts as antagonist for CXCL12/SDF-1, a chemokine involved in tumor growth.

Limitations

While the high-selectivity and tight-binding of RNA aptamers have generated interest in their use as pharmaceuticals, there are many problems which have prevented them from being successful in vivo. For one, without modifications RNA aptamers are degraded after being introduced into the body by nucleases in the span of a few minutes. Also, due to their small size, RNA aptamers can be removed from the bloodstream by the renal system. Because of their negative charge, RNA aptamers are additionally known to bind proteins in the bloodstream, leading to non-target tissue delivery and toxicity. Care must also be taken when isolating the RNA aptamers, as aptamers which contain repeated Cytosine-Phosphate-Guanine (CpG) sequences will cause immune system activation through the Toll-like receptor pathway.

In order to combat some of the in vivo limitations of RNA aptamers, various modifications can be added to the nucleotides to aid in efficacy of the aptamer. For instance, a polyethylene glycol (PEG) moiety can be attached to increase the size of the aptamer, thereby preventing its removal from the bloodstream by the renal glomerulus. However, PEG has been implicated in allergic reactions during in vivo testing. Furthermore, modifications can be added to prevent nuclease degradation, such as a 2’ fluoro or amino group as well as a 3’ inverted thymidine. Additionally, the aptamer can be synthesized so that the ribose sugar is in the L-form instead of the D-form, further preventing nuclease recognition. Such aptamers are known as Spiegelmers. In order to prevent Toll-like receptor pathway activation, the cytosine nucleobases within the aptamer can be methylated. Nevertheless, despite these potential solutions to reduced in vivo efficacy, it is possible that chemically modifying the aptamer may weaken its binding affinity towards its target.