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61-772: This article discusses only the Morpholino antisense oligomers, which are nucleic acid analogs . The word "Morpholino" can occur in other chemical names, referring to chemicals containing a six-membered morpholine ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos "Morpholino" is often capitalized as a trade name , but this usage is not consistent across scientific literature. Morpholino oligos are sometimes referred to as PMO (for phosphorodiamidate morpholino oligomer), especially in medical literature. Vivo-Morpholinos and PPMO are modified forms of Morpholinos with chemical groups covalently attached to facilitate entry into cells. Gene knockdown

122-412: A phosphate backbone, a pentose sugar, either ribose or deoxyribose , and one of four nucleobases . An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA , which affect

183-510: A (d5SICS–dNaM) complex mimicking the natural (dG–dC) base pair. One of the most common base analogs is 5-bromouracil (5BU), the abnormal base found in the mutagenic nucleotide analog BrdU. When a nucleotide containing 5-bromouracil is incorporated into the DNA, it is most likely to pair with adenine; however, it can spontaneously shift into another isomer which pairs with a different nucleobase , guanine . If this happens during DNA replication,

244-756: A DNA duplex is the hope to obtain nanoscopic self-assembling metal wires, though this has not been realized yet. An unnatural base pair (UBP) is a designed subunit (or nucleobase ) of DNA that is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team had designed two unnatural base pairs named d5SICS and dNaM . More technically, these artificial nucleotides bearing hydrophobic nucleobases feature two fused aromatic rings that form

305-433: A connected metal-base pair. This motif does not accommodate stacked Hg in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation. Two thymines across from each other do not form a Watson-Crick base pair in a duplex; this is an example where a Watson-Crick basepair mismatch is stabilized by the formation of the metal-base pair. Another example of a metal complexing to natural nucleobases

366-423: A consequence of the loss of the targeted protein and not a consequence of the knockdown oligo type. It appears that these effects are sequence-specific; as in most cases, if a Morpholino is associated with non-target effects, the 4-base mismatch Morpholino will not trigger these effects. A cause for concern in the use of Morpholinos is the potential for "off-target" effects. Whether an observed morphant phenotype

427-410: A d5SICS–dNaM complex or base pair in DNA. In 2014, the same team reported that they had synthesized a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed and inserted it into cells of the common bacterium E. coli , which successfully replicated the unnatural base pairs through multiple generations. This is the first known example of

488-488: A flexible arm, presumably extruding from the major groove of the helix. Due to low processivity of the nucleotides linked to bulky adducts such as florophores by [Taq polymerase]s, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling): Fluorophores find a variety of uses in medicine and biochemistry. The most commonly used and commercially available fluorescent base analogue, 2-aminopurine (2-AP), has

549-734: A fluorescence quantum yield of approximately 0.2 both in single- and in double-strands irrespective of surrounding bases. Also the oxo-homologue of tC called tC (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems. However, it is somewhat sensitive to surrounding bases in single-strands (quantum yields of 0.14–0.41). The high and stable quantum yields of these base analogues make them very bright, and, in combination with their good base analogue properties (leaves DNA structure and stability next to unperturbed), they are especially useful in fluorescence anisotropy and FRET measurements, areas where other fluorescent base analogues are less accurate. Also, in

610-461: A general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA. Morpholinos have become a standard knockdown tool in animal embryonic systems, which have a broader range of gene expression than adult cells and can be strongly affected by an off-target interaction. Following initial injections into frog or fish embryos at the single-cell or few-cell stages, Morpholino effects can be measured up to five days later, after most of

671-412: A guanine will be inserted as the opposite base analog, and in the next DNA replication, that guanine will pair with a cytosine. This results in a change in one base pair of DNA, specifically a transition mutation . Additionally, nitrous acid (HNO2) is a potent mutagen that acts on replicating and non-replicating DNA. It can cause deamination of the amino groups of adenine, guanine and cytosine. Adenine

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732-514: A high-fluorescence quantum yield free in solution (0.68) that is considerably reduced (appr. 100 times but highly dependent on base sequence) when incorporated into nucleic acids. The emission sensitivity of 2-AP to immediate surroundings is shared by other promising and useful fluorescent base analogues like 3-MI, 6-MI, 6-MAP, pyrrolo-dC (also commercially available), modified and improved derivatives of pyrrolo-dC, furan-modified bases and many other ones (see recent reviews). This sensitivity to

793-548: A journal article and in book form. Morpholinos are in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and genetic diseases . A Morpholino-based drug eteplirsen from Sarepta Therapeutics received accelerated approval from the US Food and Drug Administration in September 2016 for the treatment of some mutations causing Duchenne muscular dystrophy , although

854-462: A living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM. The successful incorporation of

915-422: A methyl-7-guanosine), and several bases of rRNAs (methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in or near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine). Other common tRNA base modifications are pseudouridine (which gives its name to

976-414: A mutant strain (though compensation will obscure a phenotype in some mutants), by testing the Morpholino in a null mutant background to detect additional phenotypic changes or by dominant-negative methods. As mentioned above, rescue of observed phenotypes by coinjecting a rescue mRNA is, when feasible, a reliable test of specificity of a Morpholino. For a Morpholino to be effective, it must be delivered past

1037-473: A sequence of bases in DNA , is converted into the structure of a protein. A Morpholino can modify splicing, block translation, or block other functional sites on RNA depending on the Morpholino's base sequence. Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of

1098-526: A specific metal. For example, a nucleoside modified with a pyridine-2,6-dicarboxylate has shown to bind tightly to Cu , whereas other divalent ions are only loosely bound. The tridentate character contributes to this selectivity. The fourth coordination site on the copper is saturated by an oppositely arranged pyridine nucleobase. The asymmetric metal base pairing system is orthogonal to the Watson-Crick base pairs. Another example of an artificial nucleobase

1159-648: A target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA. Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo . Developmental biologists inject Morpholino oligos into eggs or embryos of zebrafish , African clawed frog ( Xenopus ), sea urchin and killifish ( F. heteroclitus ) producing morphant embryos, or electroporate Morpholinos into chick embryos at later development stages. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture . Vivo-Morpholinos, in which

1220-654: A third base pair for replication and transcription. Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins. The possibility has been proposed and studied, both theoretically and experimentally, of implementing an orthogonal system inside cells independent of

1281-630: A third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins . Earlier, the artificial strings of DNA did not encode for anything, but scientists speculated they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses. Transcription of DNA containing unnatural base pairs and translation of corresponding mRNA were actually achieved recently. In November 2017,

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1342-797: Is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called tubercidin , an antibiotic. It has been suggested that the RNA world may have been preceded by an "RNA-like world" where other nucleic acids with a different backbone, such as GNA , PNA , and TNA existed, however, evidence for this hypothesis been called "tenuous". Naturally occurring bases can be divided into two classes according to their structure: Artificial nucleotides ( Unnatural Base Pairs (UBPs) named d5SICS UBP and dNaM UBP ) have been inserted into bacterial DNA but these genes did not template mRNA or induce protein synthesis. The artificial nucleotides featured two fused aromatic rings which formed

1403-426: Is achieved by reducing the expression of a particular gene in a cell. In the case of protein-coding genes, this usually leads to a reduction in the quantity of the corresponding protein in the cell. Knocking down gene expression is a method for learning about the function of a particular protein; in a similar manner, causing a specific exon to be spliced out of the RNA transcript encoding a protein can help to determine

1464-406: Is deaminated to hypoxanthine , which base pairs to cytosine instead of thymine. Cytosine is deaminated to uracil, which base pairs with adenine instead of guanine. Deamination of guanine is not mutagenic. Nitrous acid-induced mutations also are induced to mutate back to wild-type. Commonly fluorophores (such as rhodamine or fluorescein ) are linked to the ring linked to the sugar (in para) via

1525-681: Is debated, but there are several unused possibilities. Furthermore, adenine is not the most stable choice for base pairing: in Cyanophage S-2L, diaminopurine (DAP) is used instead of adenine. Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (weak A-T vs strong C-G). This improved stability affects protein-binding interactions that rely on those differences. Other combination include: However, correct DNA structure can form even when

1586-401: Is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. However, the polyelectrolyte theory of the gene proposes that a genetic molecule require a charged backbone to function. In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides in

1647-422: Is due to the intended knockdown or an interaction with an off-target RNA can often be addressed in embryos by running another experiment to confirm that the observed morphant phenotype results from the knockdown of the expected target. This can be done by recapitulating the morphant phenotype with a second, non-overlapping Morpholino targeting the same mRNA, by confirmation of the observed phenotypes by comparing with

1708-418: Is joined there by various other eukaryotic initiation factors , forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon , and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene , encoded as

1769-446: Is that with hydroxypyridone nucleobases, which are able to bind Cu inside the DNA duplex. Five consecutive copper-hydroxypyridone base pairs were incorporated into a double strand, which were flanked by only one natural nucleobase on both ends. EPR data showed that the distance between copper centers was estimated to be 3.7 ± 0.1 Å, while a natural B-type DNA duplex is only slightly larger (3.4 Å). The appeal for stacking metal ions inside

1830-429: Is the formation of A-Zn-T and G-Zn-C at high pH; Co and Ni also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated. A large variety of artificial nucleobases have been developed for use as metal base pairs. These modified nucleobases exhibit tunable electronic properties, sizes, and binding affinities that can be optimized for

1891-682: Is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including uptake into muscle cells with Duchenne muscular dystrophy or the vascular endothelial cells stressed during balloon angioplasty ). Though they permeate through intercellular spaces in tissues effectively, unconjugated PMOs have limited distribution into the cytosol and nuclear spaces within healthy tissues following IV administration. Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with cell-penetrating peptides , and, while toxicity has been associated with moderate doses of

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1952-769: The TΨC loop ), dihydrouridine (which does not stack as it is not aromatic), queuosine, wyosine, and so forth. Nevertheless, these are all modifications to normal bases and are not placed by a polymerase. Canonical bases may have either a carbonyl or an amine group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to base pair (Watson-Crick base pairing) via hydrogen bonds (amine with ketone, purine with pyrimidine). Adenine and 2-aminoadenine have one/two amine group(s), whereas thymine has two carbonyl groups, and cytosine and guanine are mixed amine and carbonyl (inverted in respect to each other). The precise reason why there are only four nucleotides

2013-472: The cell membrane into the cytosol of a cell. Once in the cytosol, Morpholinos freely diffuse between the cytosol and nucleus, as demonstrated by the nuclear splice-modifying activity of Morpholinos observed after microinjection into the cytosol of cells. Different methods are used for delivery into embryos, into cultured cells or into adult animals. A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at

2074-425: The central nervous system and somite tissues of zebrafish embryos. Most of these effects are due to activation of p53 -mediated apoptosis and can be suppressed by co-injection of an anti-p53 Morpholino along with the experimental Morpholino. Moreover, the p53-mediated apoptotic effect of a Morpholino knockdown has been phenocopied using another antisense structural type, showing the p53-mediated apoptosis to be

2135-427: The 3' hydroxyl group normally present in DNA and therefore cannot bond with the next base. The lack of the 3' hydroxyl group terminates the chain reaction as the DNA polymerases mistake it for a regular deoxyribonucleotide. Another chain-terminating analogue that lacks a 3' hydroxyl and mimics adenosine is called cordycepin . Cordycepin is an anticancer drug that targets RNA replication. Another analogue in sequencing

2196-462: The Watson-Crick hydrogen bonds are replaced by the interaction between a metal ion with nucleosides acting as ligands. The possible geometries of the metal that would allow for duplex formation with two bidentate nucleosides around a central metal atom are tetrahedral , dodecahedral , and square planar . Metal-complexing with DNA can occur by the formation of non-canonical base pairs from natural nucleobases with participation by metal ions and also by

2257-463: The approval process was mired in controversy. Other Morpholino-based drugs golodirsen , viltolarsen , and casimersen (also for Duchenne muscular dystrophy) were approved by the FDA in 2019–2021. Morpholino oligos were conceived by Summerton ( Gene Tools ) at AntiVirals Inc. (now Sarepta Therapeutics) and originally developed in collaboration with Weller. Morpholinos are synthetic molecules that are

2318-551: The bacteria to synthesize "unnatural" proteins. Another demonstration of UBPs were achieved by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in vitro in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as

2379-424: The bases are not paired via hydrogen bonding; that is, the bases pair thanks to hydrophobicity, as studies have shown with DNA isosteres (analogues with same number of atoms) such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). An alternative hydrophobic pair could be isoquinoline and pyrrolo[2,3-b]pyridine Other noteworthy basepairs: In metal base-pairing,

2440-452: The borders of introns on a strand of pre-mRNA, or by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure, or by interfering with the binding of splice regulatory proteins such as splice silencers and splice enhancers . Preventing the binding of snRNP U1 (at the donor site) or U2 / U5 (at the polypyrimidine moiety and acceptor site) can cause modified splicing , commonly excluding exons from

2501-444: The coding region of the targeted transcript (called " knocking down " gene expression ). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins,

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2562-651: The culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides. The artificial nucleotides featured 2 fused aromatic rings. Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canonical bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes. Nucleic acid analogues are used in molecular biology for several purposes: Ribose 's 2' hydroxy group reacts with

2623-464: The exchanging the hydrogen atoms that are part of the Watson-Crick base pairing by metal ions. Introduction of metal ions into a DNA duplex has shown to have potential magnetic or conducting properties, as well as increased stability. Metal complexing has been shown to occur between natural nucleobases . A well-documented example is the formation of T-Hg-T, which involves two deprotonated thymine nucleobases that are brought together by Hg and forms

2684-474: The function of the protein moiety encoded by that exon or can sometimes knock down the protein activity altogether. These molecules have been applied to studies in several model organisms , including mice , zebrafish , frogs and sea urchins . Morpholinos can also modify the splicing of pre-mRNA or inhibit the maturation and activity of miRNA. Techniques for targeting Morpholinos to RNAs and delivering Morpholinos into cells have recently been reviewed in

2745-1013: The mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions. Targets of U11 / U12 snRNPs can also be blocked. Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction ( RT-PCR ) and is seen as a band shift after gel electrophoresis of RT-PCR products. Morpholinos have been used to block miRNA activity and maturation. Fluorescein -tagged Morpholinos combined with fluorescein-specific antibodies can be used as probes for in-situ hybridization to miRNAs. Morpholinos can block ribozyme activity. U2 and U12 snRNP functions have been inhibited by Morpholinos. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts . Morpholinos can block RNA editing, poly(A) tailing and translocation sequences. Morpholino activities against this variety of targets suggest that Morpholinos can be used as

2806-435: The microenvironment has been utilized in studies of e.g. structure and dynamics within both DNA and RNA, dynamics and kinetics of DNA-protein interaction and electron transfer within DNA. A newly developed and very interesting group of fluorescent base analogues that has a fluorescence quantum yield that is nearly insensitive to their immediate surroundings is the tricyclic cytosine family. 1,3-Diaza-2-oxophenothiazine, tC, has

2867-404: The oligo is covalently linked to a delivery dendrimer , enter cells when administered systemically in adult animals or in tissue cultures. In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm . The small subunit of the ribosome usually starts by binding at the 5' end of the mRNA and

2928-719: The peptide conjugates, they have been used in vivo for effective oligo delivery at doses below those causing observed toxicity. An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol. Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases. Nucleic acid analogues Nucleic acid analogues are compounds which are analogous (structurally similar) to naturally occurring RNA and DNA , used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts:

2989-443: The phosphate linked 3' hydroxy group, making RNA too unstable to be used or synthesized reliably. To overcome this, a ribose analogue can be used. The most common RNA analogues are 2'-O-methyl-substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino , and peptide nucleic acid ( PNA ). Although these oligonucleotides have a different backbone sugar—or, in the case of PNA, an amino acid residue in place of

3050-417: The processes of organogenesis and differentiation are past, with observed phenotypes consistent with target-gene knockdown. Control oligos with irrelevant sequences usually produce no change in embryonic phenotype, evidence of the Morpholino oligo's sequence-specificity and lack of non-antisense effects. The dose required for a knockdown can be reduced by coinjection of several Morpholino oligos targeting

3111-468: The product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing . In terms of structure, the difference between Morpholinos and DNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylene morpholine rings linked through phosphorodiamidate groups instead of phosphates . The figure compares

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3172-492: The properties of the chain (PNA can even form a triple helix ). Nucleic acid analogues are also called xeno nucleic acids and represent one of the main pillars of xenobiology , the design of new-to-nature forms of life based on alternative biochemistries. Artificial nucleic acids include peptide nucleic acids (PNA), Morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) and hexitol nucleic acids (HNA). Each of these

3233-688: The rescue RNA makes recovery of the wild-type phenotype impossible. In embryos, Morpholinos can be tested in null mutants to check for unexpected RNA interactions, then used in a wild-type embryo to reveal the acute knockdown phenotype. The knockdown phenotype is often more extreme than the mutant phenotype; in the mutant, effects of losing the null gene can be concealed by genetic compensation. Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade Morpholinos, nor are they degraded in serum or in cells. Up to 18% of Morpholinos appear to induce nontarget-related phenotypes including cell death in

3294-469: The rescue mRNA contains no target for the Morpholino. The rescue mRNA's coding region encodes the protein of interest. Translation of the rescue mRNA replaces production of the protein that was knocked down by the Morpholino. Since the rescue mRNA would not affect phenotypic changes due to the Morpholino's off-target gene expression modulation, this return to wild-type phenotype is further evidence of Morpholino specificity. In some cases, ectopic expression of

3355-436: The ribose phosphate—they still bind to RNA or DNA according to Watson and Crick pairing while being immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using the phosphoramidite strategy or, for PNA, other methods of peptide synthesis . Dideoxynucleotides are used in sequencing . These nucleoside triphosphates possess a non-canonical sugar, dideoxyribose, which lacks

3416-471: The same family of cytosine analogues, a FRET-acceptor base analogue, tC nitro , has been developed. Together with tC as a FRET-donor this constitutes the first nucleic acid base analogue FRET-pair ever developed. The tC-family has, for example, been used in studies related to polymerase DNA-binding and DNA-polymerization mechanisms. In a cell, there are several non-canonical bases present: CpG islands in DNA (often methylated), all eukaryotic mRNA (capped with

3477-444: The same mRNA, which is an effective strategy for reducing or eliminating dose-dependent off-target RNA interactions. mRNA rescue experiments can sometimes restore the wild-type phenotype to the embryos and provide evidence for the specificity of a Morpholino. In an mRNA rescue, a Morpholino is co-injected with an mRNA that codes for the morphlino's protein. However, the rescue mRNA has a modified 5'-UTR (untranslated region) so that

3538-571: The same team at the Scripps Research Institute that first introduced two extra nucleobases into bacterial DNA reported having constructed a semi-synthetic E. coli bacteria able to make proteins using such DNA. Its DNA contained six different nucleobases : four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). The bacteria had two corresponding RNA bases included in two new codons, additional tRNAs recognizing these new codons (these tRNAs also contained two new RNA bases within their anticodons) and additional amino acids, enabling

3599-663: The single-cell or few-cell stage; an alternative method for embryonic delivery is electroporation , which can deliver oligos into tissues of later embryonic stages. Common techniques for delivery into cultured cells include the Endo-Porter peptide (which causes the Morpholino to be released from endosomes ), the Special Delivery system (no longer commercially available, used a Morpholino-DNA heteroduplex and an ethoxylated polyethylenimine delivery reagent), electroporation, or scrape loading. Delivery into adult tissues

3660-580: The structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits. Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates , siRNA ). Instead, Morpholinos act by "steric blocking", binding to

3721-444: The targeted proteins become undetectable by Western blot . In 2016 a synthetic peptide-conjugated PMO (PPMO) was found to inhibit the expression of New Delhi Metallo-beta-lactamase , an enzyme that many drug-resistant bacteria use to destroy carbapenems. Morpholinos can interfere with pre-mRNA processing steps either by preventing splice-directing small nuclear ribonucleoproteins ( snRNP ) complexes from binding to their targets at

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