Morpholino - AbsoluteAstronomy.com

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

, a Morpholino is a molecule

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

in a particular structural

Molecular structure

The molecular structure of a substance is described by the combination of nuclei and electrons that comprise its constitute molecules. This includes the molecular geometry , the electronic properties of the...

family that is used to modify gene expression

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

. Morpholino oligomer

Oligomer

In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...

s (oligos) are an antisense technology used to block access of other molecules to specific sequences within nucleic acid

Nucleic acid

Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid (RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

).

This article discusses only the Morpholino antisense oligos, which are nucleic acid analogs

Nucleic acid analogues

Nucleic acid analogues are compounds structurally similar to naturally occurring RNA and DNA, used in medicine and in molecular biology research....

. The word "morpholino" can occur in other chemical names, referring to chemicals containing a six-membered morpholine

Morpholine

Morpholine is an organic chemical compound having the chemical formula O2NH. This heterocycle, pictured at right, features both amine and ether functional groups. Because of the amine, morpholine is a base; its conjugate acid is called morpholinium...

ring. To help avoid confusion with other morpholine-containing molecules, when describing oligos "Morpholino" is often capitalized as a trade name

Trade name

A trade name, also known as a trading name or a business name, is the name which a business trades under for commercial purposes, although its registered, legal name, used for contracts and other formal situations, may be another....

, though this usage is not consistent throughout the scientific literature. Morpholinos are sometimes referred to as "PMO" (phosphorodiamidate morpholino oligo).

Morpholinos are usually used as a research tool for reverse genetics

Reverse genetics

Reverse genetics is an approach to discovering the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics...

by knocking down

Gene knockdown

Gene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide with a sequence complementary to either an mRNA transcript or a gene...

gene function. This is achieved by preventing cells from making a targeted protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

or by modifying the splicing

Splicing (genetics)

In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...

of pre-mRNA. Knocking down gene expression is a powerful method for learning about the function of a particular protein; in similar manner, causing a specific exon

Exon

An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

to be spliced out of a protein can help to determine the function of the protein moiety encoded

Genetic code

The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....

by that exon. These molecules have been applied to studies in several model organism

Model organism

A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...

s, including mice

Mouse

A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...

, zebrafish, frog

Xenopus

Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa. There are 19 species in the Xenopus genus...

s, and sea urchin

Sea urchin

Sea urchins or urchins are small, spiny, globular animals which, with their close kin, such as sand dollars, constitute the class Echinoidea of the echinoderm phylum. They inhabit all oceans. Their shell, or "test", is round and spiny, typically from across. Common colors include black and dull...

s.

Morpholinos are also in development as pharmaceutical

Pharmacology

Pharmacology is the branch of medicine and biology concerned with the study of drug action. More specifically, it is the study of the interactions that occur between a living organism and chemicals that affect normal or abnormal biochemical function...

therapeutics targeted against pathogen

Pathogen

A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...

ic organisms such as bacteria

Bacteria

Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

or virus

Virus

A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

es and for amelioration of genetic diseases

Genetic disorder

A genetic disorder is an illness caused by abnormalities in genes or chromosomes, especially a condition that is present from before birth. Most genetic disorders are quite rare and affect one person in every several thousands or millions....

. These synthetic oligos were conceived by James E. Summerton (Gene Tools, LLC

Gene Tools

Gene Tools, LLC is a limited liability company located in Philomath, Oregon, United States that manufactures Morpholino antisense oligos and delivery reagents. Gene Tools was founded in 1997 and began regularly shipping custom-sequence Morpholino oligos in 2000. The manager and general partner, Dr...

) and developed in collaboration with Dwight D. Weller (AVI BioPharma Inc.

AVI Bio Pharma

AVI BioPharma Inc. is a medical research and drug development company with corporate offices and research facility in Bothell, Washington, United States. Incorporated in 1980, the company maintains some laboratory capability in Corvallis, Oregon...

Structure

Morpholinos are synthetic

Oligonucleotide synthesis

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...

molecule

Molecule

A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s that are the product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA by standard nucleic acid base-pairing

Base pair

In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...

. Structurally, the difference between Morpholinos and DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine

Morpholine

rings instead of deoxyribose

Deoxyribose

Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...

rings and linked through phosphorodiamidate groups instead of phosphates. This may be easiest to visualize by referring to the first figure and comparing the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic

Ion

An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...

phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH

In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits. Morpholinos are most commonly used as single-stranded oligos, though heteroduplex

Heteroduplex

A heteroduplex is a double-stranded molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from different sources, such as from different homologous chromosomes or even from different organisms....

es of a Morpholino strand and a complementary

Complementarity (molecular biology)

In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...

DNA strand may be used in combination with cationic cytosol

Cytosol

The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....

ic delivery reagents.

Function

Morpholinos do not degrade their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA

Sírna

Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...

). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA and simply getting in the way of 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

Xenopus

Xenopus is a genus of highly aquatic frogs native to Sub-Saharan Africa. There are 19 species in the Xenopus genus...

), sea urchin

Sea urchin

, and killifish (F. heteroclitus) producing morphant

Morphant

An organism which has been treated with a Morpholino antisense oligo to temporarily knock down expression of a targeted gene is called a Morphant.-Background:...

embryos, or electroporate Morpholinos into chick

Chicken

The chicken is a domesticated fowl, a subspecies of the Red Junglefowl. As one of the most common and widespread domestic animals, and with a population of more than 24 billion in 2003, there are more chickens in the world than any other species of bird...

embryos at later stages of development. With appropriate cytosolic delivery systems, Morpholinos are effective in cell culture

Cell culture

Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...

. Morpholinos are being developed as pharmaceuticals under the name "NeuGenes" by AVI BioPharma Inc. They have been used in mammals such as mice

Mouse

and some are currently being tested in clinical trial

Clinical trial

Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...

s for treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy

Duchenne muscular dystrophy is a recessive X-linked form of muscular dystrophy, which results in muscle degeneration, difficulty walking, breathing, and death. The incidence is 1 in 3,000 boys. Females and males are affected, though females are rarely affected and are more often carriers...

in human

Human

Humans are the only living species in the Homo genus...

Normal gene expression in eukaryotes

In eukaryotic

Eukaryote

A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

organisms, pre-mRNA is transcribed

Transcription (genetics)

Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

in the nucleus, introns are spliced

Splicing (genetics)

out, then the mature mRNA is exported from the nucleus

Cell nucleus

In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...

to the cytoplasm

Cytoplasm

The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

. The small subunit of the ribosome

Ribosome

A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....

usually starts by binding to one end of the mRNA and is joined there by various other eukaryotic initiation factor

Eukaryotic initiation factor

Eukaryotic initiation factors are proteins involved in the initiation phase of eukaryotic translation. They function in forming a complex with the 40S ribosomal subunit and Met-tRNAi called the 43S preinitation complex , recognizing the 5' cap structure of mRNA and recruiting the 43S PIC to mRNA,...

s, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon

Start codon

The start codon is generally defined as the point, sequence, at which a ribosome begins to translate a sequence of RNA into amino acids.When an RNA transcript is "read" from the 5' carbon to the 3' carbon by the ribosome the start codon is the first codon on which the tRNA bound to Met,...

, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein

Protein

begins. This entire process is referred to as gene expression; it is the process by which the information in a gene

Gene

A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

, encoded as a sequence of bases in DNA

DNA

, is converted into the structure of a protein. A Morpholino can modify splicing or block translation, depending on the Morpholino's base sequence.

Blocking translation

Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal

Ribosome

A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....

initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript

Transcription (genetics)

(called "knocking down

Gene knockdown

" gene expression

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, the targeted proteins become undetectable by Western blot

Western blot

The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...

(e.g., figure 1 in:).

Modifying pre-mRNA splicing

Morpholinos can interfere with pre-mRNA processing steps either by preventing splice-directing small nuclear ribonucleoproteins (snRNP

SnRNP

snRNPs , or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs...

) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA, or by blocking the nucleophilic

Nucleophile

A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...

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

Exonic splicing enhancer

An exonic splicing enhancer is a DNA sequence motif consisting of 6 bases within an exon that directs, or enhances, accurate splicing of hetero-nuclear RNA or pre-mRNA into messenger RNA .- The Basics :...

. Preventing the binding of snRNP U1 (at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptor site) can cause modified splicing

Splicing (genetics)

, commonly excluding exon

Exon

s from the mature mRNA. Targeting some splice targets results in intron

Intron

An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...

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

Gel electrophoresis

Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

of RT-PCR products.

Blocking other mRNA sites

Morpholinos have been used to block miRNA

Mirna

Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...

activity and maturation. They can also block ribozyme

Ribozyme

A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes catalyze either the hydrolysis of one of their own...

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

Frameshift mutation

A frameshift mutation is a genetic mutation caused by indels of a number of nucleotides that is not evenly divisible by three from a DNA sequence...

. Activities of Morpholinos against this variety of targets suggest that Morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.

Specificity, stability and non-antisense effects

Morpholinos have become a standard knockdown tool in animal embryo

Embryo

An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...

nic systems, which have a broader range of gene expression than adult cell

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

s 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 the processes of organogenesis

Organogenesis

In animal development, organogenesis is the process by which the ectoderm, endoderm, and mesoderm develop into the internal organs of the organism. Internal organs initiate development in humans within the 3rd to 8th weeks in utero...

and differentiation

Cellular differentiation

In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...

are past, with observed phenotype

Phenotype

A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...

s 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 the same mRNA, which is an effective strategy for reducing or eliminating dose-dependent off-target RNA interactions.

mRNA rescue experiments can often 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 same protein that the Morphlino is targeted to knock down. However, the rescue mRNA has a modified 5'-UTR

Five prime untranslated region

A messenger ribonucleic acid molecule codes for a protein through translation. The mRNA also contains regions that are not translated: in eukaryotes these include the 5' untranslated region, 3' untranslated region, 5' cap and poly-A tail....

(untranslated region) so that the rescue mRNA contains no target for the Morpholino but the rescue mRNA's coding region

Coding region

The coding region of a gene, also known as the coding sequence or CDS, is that portion of a gene's DNA or RNA, composed of exons, that codes for protein. The region is bounded nearer the 5' end by a start codon and nearer the 3' end with a stop codon...

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 modulation of off-target gene expression by the Morpholino, this return to wild-type phenotype is further evidence of Morpholino specificity.

Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nuclease

Nuclease

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....

s do not degrade Morpholinos, nor are they degraded in serum or in cells. Morpholinos do not activate toll-like receptor

Toll-like receptor

Toll-like receptors are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors that recognize structurally conserved molecules derived from microbes...

s and so they do not activate innate immune responses

Innate immune system

The innate immune system, also known as non-specific immune system and secondary line of defence, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner...

such as interferon

Interferon

Interferons are proteins made and released by host cells in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.IFNs belong to...

induction or the NF-κB mediated inflammation

Inflammation

Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process...

response. Morpholinos are not known to modify methylation of DNA

DNA methylation

DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...

.

A cause for concern in the use of Morpholinos is the potential for "off-target" effects. Up to 18% of Morpholinos appear to have nontarget-related phenotypes including cell death in the central nervous system and somite

Somite

A somite is a division of the body of an animal. In vertebrates this is mainly discernible in the embryo stage; in arthropods it is a characteristic of a hypothetical ancestor.- In vertebrates :...

tissues of zebrafish embryos. Most of these effects have been shown to be due to activation of p53

P53

p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...

-mediated apoptosis

Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

, 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 a consequence of the loss of the targeted protein and not a consequence of the oligo type used to knock down the protein. 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. The question of whether an observed morphant

Morphant

An organism which has been treated with a Morpholino antisense oligo to temporarily knock down expression of a targeted gene is called a Morphant.-Background:...

phenotype is due to the intended knockdown or an off-target interaction can often be addressed 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 or by confirmation of the observed phenotypes by use of a mutant strain or 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.

Delivery

For a Morpholino to be effective, it must be delivered past the cell membrane

Cell membrane

The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...

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

Microinjection

Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the...

apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage; an alternative method for embryonic delivery is electroporation

Electroporation

Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...

, 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 endosome

Endosome

In biology, an endosome is a membrane-bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for...

s), the Special Delivery system (using a Morpholino-DNA heteroduplex

Heteroduplex

and an ethoxylated polyethylenimine

Polyethylenimine

Linear polyethyleneimines contain all secondary amines, in contrast to branched PEIs which contain primary, secondary and tertiary amino groups. The linear PEIs are solids at room temperature where branched PEIs are liquids at all molecular weights. Linear polyethyleneimines are soluble in hot...

delivery reagent), electroporation, or scrape loading.

Delivery into adult tissues

Tissue (biology)

Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...

is usually difficult, though there are a few systems allowing useful uptake of unmodified Morpholino oligos (including the inherently leaky muscle

Muscle

Muscle is a contractile tissue of animals and is derived from the mesodermal layer of embryonic germ cells. Muscle cells contain contractile filaments that move past each other and change the size of the cell. They are classified as skeletal, cardiac, or smooth muscles. Their function is to...

cells caused by Duchenne muscular dystrophy

Duchenne muscular dystrophy

or the vascular endothelial

Endothelium

The endothelium is the thin layer of cells that lines the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. These cells are called endothelial cells. Endothelial cells line the entire circulatory system, from the heart...

cells stressed during balloon angioplasty

Angioplasty

Angioplasty is the technique of mechanically widening a narrowed or obstructed blood vessel, the latter typically being a result of atherosclerosis. An empty and collapsed balloon on a guide wire, known as a balloon catheter, is passed into the narrowed locations and then inflated to a fixed size...

). 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 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.

Structure

Function

Normal gene expression in eukaryotes

Blocking translation

Modifying pre-mRNA splicing

Blocking other mRNA sites

Specificity, stability and non-antisense effects

Delivery

Further reading