Saturday, July 30, 2011
Thursday, July 28, 2011
FERTILIZATION
- Xulkifal yousaf [me_the_charlie@hotmail.com] Fertilization
- FERTILIZATION
- Recognition of Egg and Sperm: 5 steps, (1). Chemo attraction (2). Release of acrosomal enzymes (3). Binding of sperm to extra cellular envelopes (4). Passage through extra cellular envelopes (5). Fusion of sperm and egg nuclei (2 & 3 can be reversed).
- FERTILIZATION
- Sperm attraction…..species specific in many invertebrates, chemo taxis, also timings in these animals. In sea urchin a peptide resact is such chemo tactic molecule. It is also sperm activating molecule.
- Egg jelly has other compounds as well, which bind with specific receptors on sperm, which inturn opens calcium ion channels, leading to exocytosis of acrosome.
- FERTILIZATION
- Acrosome reaction: In marine invertebrates, fusion of acrosomal membrane with sperm membrane & the formation of acrosomal process. Release of proteolytic enzymes ->-> go through jelly coats ->->egg membrane.
- Transportation of sperm and its capacitation. Active, also passive through contraction, species specific recognition (as in sea urchin).
- FERTILIZATION
- Acrosomal protein is Bindin….present on acrosomal process…which is species specific. Its specific receptors not all over the egg membrane……so limited sites of contact and penetration (first step towards block to polyspermy).
- (In mammals, zona pellucida has many glycoproteins ….ZP-1, ZP-2, ZP-3….it is ZP-3 which affects binding & it also triggers acrosomal reaction.
- FERTILIZATION
- ZP-3 of ZP of egg crosslinks with a sperm protein called galactosyltransferase-Ι(which binds with carbohydrate residue of ZP-3)
- Exocytosis of acrosome (its breakdown) releases many proteases----which make a hole in ZP; secondary binding of spermatozoa is through ZP-2).
- FERTILIZATION
- Gamete Fusion and Prevention Of Polyspermy : Fusion of sperm and egg membranes ->->polymerization of actin in egg->-> fertilization cone, as does the sperm actin which forms the acrosomal process.
- These two projections meet so as to form a continuous bridge through which sperm nucleus & centiole pass.
- FERTILIZATION
- (In mammals sperms attach to egg membrane at a side of head (equatorial domain)….of head).
- Prevention of polyspermy: In sea urchin …fast & slow processes……fast by changing of the electric potential of egg membrane, high Na contents outside egg (in sea water), inside high K.
- FERTILIZATION
- Resting membrane potential -70 mv (inside of egg negatively charged)…within 1-3 sec. potential changes to positive, if experimentally this potential is changed polyspermy will be duly affected (i.e. by creating negative charge polyspermy can occur OR no fertilization by keeping the charge positive).
- FERTILIZATION
- Slow block, sea urchin, cortical granule reaction, 15000 granules,1 µm in diameter, fuse with plasma membrane of egg and release contents into space between it & vitelline membrane, i.e. exocytosis, several proteins released.
- First is cortical granule serine protease, it dissolves protein part that connect vitelline envelope protein to cell membrane, also clips off bindin receptors and any sperm attached to them.
- FERTILIZATION
- Another mucopolysaccharide causes formation of fertilization envelope (by causing water get into the space between plasmalemma & vitelline membrane).
- A third protein released by cortical granules is peroxidase enzyme, which hardens fertilization envelope by cross linking tyrosine residues on adjacent proteins. This starts at sperm entry & spreads all over---- starts within 20 seconds and is finished by 60 seconds.
- FERTILIZATION
- A 4th protein, hyalin, forms a coating around egg i.e. a hyalin layer, similar in mammalian egg but no fertilization membrane is formed, some other peculiar proteins in mouse i.e. ZP-3 & ZP-2 are knocked out.
- Calcium as initiator of cortical reaction : Free calcium increases a lot. No influx but release from within egg i.e. intra cellular storage.
- This can be monitored visually by ca-activated luminescent dye such as aequorin or fura-2.
- FERTILIZATION
- These emit light when they bind with calcium.If injected with these dyes then fertilized, a wave of light starts from sperm entry point & go to other end ---completed within 30 sec. An ionophore A23187 (i.e. it transports calcium across membrane from outside), can cause cortical granules reaction & elevation of fertilization membrane if egg is immersed in this medium.
- FERTILIZATION
- If no calcium in surrounding water even then this reaction occurs, so it must be activating ca++ already present.
- Activation Of Egg Metabolism: Early & late responses.
- FERTILIZATION
- Early; Increase in calcium conc.->-> cell division, protein synthesis (calcium increases from 0.1 to 1.0 µM).Several waves of calcium release….. Several activities in egg related to these waves. If ca-chelating agent such as EGTA is injected ->->no cortical granule reaction, no change in membrane potential ->->no action potential.
- FERTILIZATION
- Conversely injection of calcium ionophore (A23187) ->->all activities i.e. elevation of fertilization membrane, rise in intra cellular pH, increase in O2 consumption, increase in the protein synthesis ,increase in DNA synthesis ,activation of enzyme NAD+ kinase which coverts NAD+ to NADP which is important for construction of new cell membranes , needed at cleavage.
- FERTILIZATION
- Late Responses; DNA & protein synthesis, if pH increased artificially, DNA & protein synthesis ensues, nuclear envelope breakdown. Protein synthesis…. uses mRNA ….. already synthesized, especially encoding for histone, tubulin, actins, and morphogenetic proteins. The mRNA that was masked is now released (by maskin protein).
- FERTILIZATION Fusion Of Genetic Material: In sea urchin ----♀pronucleus already haploid, ♂ pronucleus decondenses, fuse to form zygote nuclei (diploid). In mammals at fertilization ♀ nucleus at metaphase of 2nd meiotic division, calcium inactivates MAP kinase & DNA synthesis ensues. Two pronucei move towards each other: no envelope break down & chromosomes come on spindle (so true diploid nucleus is NOT formed in zygote but after first division) Fertilization May 21, 2010
- FERTILIZATION
- Rearrangement Of Egg Cytoplasm: Fertilization leads to a lot of cytoplasmic movement, crucial for later cell differentiation. Rearrangement can be seen in tunicate ( Styla)…. various colors.
- Also in amphibian cortex moves by about 30 degree towards sperm entry ->-> grey crescent exposed, this rotation is brought about by microtubules localized between cortex & inner cytoplasm.
- FERTILIZATION
- Sperm Capacitation: Newly ejaculated sperms need to remain in ♀ reproductive tract to be able to undergo acrosomal reaction.
- The set of physiological changes through which sperms become competent to fertilize an egg, is called capacitation.
- FERTILIZATION
- Such changes involve (1). Removal of cholesterol from the sperm cell membrane. (2). Some particular proteins &/or carbohydrates are also lost during capacitation. (3). The membrane potential becomes more negative, as K ions leave the sperm. (4). Protein phosphorylation also takes place during capacitation.
- Capacitation can be achieved in vitro , by incubating sperm in media containing calcium, bicarbonate and serum albumin.
MICROARRAY TECHNOLOGY
BACKGROUND: NEED FOR MICROARRAY TECHNOLOGY:
A cell functions by using its genes to produce proteins and although each cell within an organism will usually contain the same set of genes, there are significant differences in which genes are activated and how they are controlled because a fraction of these genes are turned on and it is the subset that is "expressed" that confers unique properties to each cell type. "Gene expression" is the term used to describe the transcription of the information contained within the DNA. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. For many years, study of a gene in this manner had to be done individually—looking at whether a specific gene is turned on (upregulated) or turned off (downregulated) under certain conditions. MICROARRAY is a technological advancement that has allowed scientists to study many, if not all, the genes of an organism’s at once. This high throughput method allows for the global study of changes in gene expression, giving us a complete cellular snapshot.
INTRODUCTION
Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. It is a high throughput technology that allows simultaneous detection of thousands of genes.
In formal terms a microarray can be defined as:
A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in a fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides. Microarrays are also referred to as DNA arrays, DNA chips, biochips and GeneChips.
Microarrays come up with the advantageous features as:
– Multiplexing: handling multiple samples at a time
– Parallelism: Studying Thousands of genes simultaneously
– Miniaturization: small sized chip
PRINCIPLE:
The principle of microarrays is based upon base paired hybridization probing, a technique that uses fluorescently labeled nucleic acid molecules as "mobile probes" to identify complementary molecules, and sequences that are able to base-pair with one another.
PARAMETERS DETERMINING THE NATURE OF A MICROARRAY TECHNOLOGY
PARAMETER OPTION
Probes: features arrayed on the microarray substrate that have known identity or sequence cDNA, oligonucleotides, proteins, peptide nucleic acids, small molecules, cells, tissues, and organisms
Fabrication: techniques to array probes on the microarray substrate In situ synthesis, robotic deposition
Targets: samples to be analyzed against the probes DNA, mRNA, proteins, enzymes, small molecules
Assays: principles based on which the targets are being analyzed Hybridization, electrophoresis, flow cytometry, ELISA
Signal readout: principles based on which the assay results can be detected Fluorescence , chemiluminescence, mass spectrometry, radioactivity, electrochemistry
Image processing: signal intensities of hybridized array spots are quantified from the scanning image Software for image processing
Informatics: computational tools with which the huge amount of data generated from a microarray experiment can be effectively stored and interpreted Database management system, data mining and visualization, interpretation of biological meaning
There are various types of microarrays, and to name a few, they include: DNA Microarrays, Protein Microarrays, Tissue Microarrays, Cellular Microarrays and Antibody Microarrays. Among the various types, DNA microarrays, protein microarrays and tissue microarrays are most widely used. The focus in the following sections will be on DNA microarrays.
DNA MICROARRAYS
DNA Microarray is the most widely used microarray technology. It is a type of nucleic acid-based multiplex technique involving high-density arrays of nucleic acids on glass that allows evaluating mRNA abundance of up to tens of thousands of genes simultaneously.
A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted (less than 1 mm) to that location. Through the use of highly accurate robotic spotters, over 30,000 spots can be placed on one slide, allowing molecular biologists to analyze virtually every gene present in a genome. The main advantage of microarrays is that the spots are single stranded DNA fragments that are strongly attached to the slide, allowing cellular DNA or RNA to be fluorescently labeled and laid overtop of the array. DNA or RNA in the overlaid sample will stick (through hybridization) to a complementary spot on the array, that is, Gene-A will stick to a spot composed of a Gene-A fragment. By exposing the microarray to a fluorescently labeled sample the DNA that hybridizes will be identifiable as glowing spots on the array, while the spots that have nothing hybridized to them will not be visible.
TYPES OF DNA MICROARRAYS
Two main types of commercial DNA microarrays are: oligonucleotide arrays and cDNA arrays.
1. OLIGONUCLEOTIDE ARRAYS
Oligonucleotide arrays (trademarked as a GeneChip by Affymetrix) use small 25 base pair gene fragments as the DNA probes to be spotted onto an array. These oligonucleotide probes are synthesized either in situ (on-chip) using photolithography or by conventional synthesis followed by on-chip immobilization.
APPLICATIONS OF OLIGONUCLEOTIDE ARRAYS
Oligonucelotide arrays are used for quantification of the amount of mRNA in a single sample (e.g. to determine the amount of mutated vs. non-mutated mRNA) or the comparison of two different samples hybridized to two separate arrays.
OLIGONUCLEOTIDE ARRAY TARGETS
Most array experimenters do not have access to micrograms of total RNA from their biological sample. A linear amplification strategy is highly recommended to obtain sample to be used for hybridization (targets). This technique consists of the following steps:
• First strand cDNA synthesis using reverse transcriptases;
• Generation of second strand cDNA using DNA polymerases; and
• In vitro transcription of the second strand cDNA to generate antisense RNA (aRNA) or complementary RNA (cRNA).
The probes are selected to have little cross-reactivity with other genes (targets) so that non-specific hybridization will be minimized.
2. cDNA ARRAYS
A cDNA array (also known as DNA microarray) is a different technology using the same principle; the probes in this case are larger pieces of DNA that are complementary to the genes one is interested in studying. With the cDNA probes prepared they can be mechanically spotted (robot spotting) onto a glass slide, normally in duplicate to serve as controls.
cDNA PROBES
• cDNA probes for making the array can also be generated from a commercially available cDNA library ensuring a close representation of the entire genome of an organism on the array.
• In an alternate target synthesis protocol, the target is generated by reverse transcription of the total RNA or the aRNA.
• Alternatively, PCR using specific primers can be used to amplify specific genes from genomic DNA to generate the cDNA probes.
When using cDNA spotted microarrays, two pools of RNA can be compared on the same array simultaneously. This is unlike Affymetrix GeneChip arrays, where RNA isolated from a single source is hybridized to an array or multiple arrays are required for comparison between more than one RNA samples.
DESIGN AND WORKING OF MICROARRAYS
DESIGN OF MICROARRAYS
Micro-array probes are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization.
• Micro-arrays are printed in situ (on chip) using a combination of photolithography and combinatorial chemistry.
• For on-chip immobilization, probes can be mechanically spotted onto a glass slide by using robot.
PHOTOLITHOGRAPHY:
The production begins with a 5-inch square of quartz wafer.
This wafer is washed with a blocking compound that can be removed by exposure to light. What is needed next is referred to as a ‘mask’, which is designed with 18-20 micron square windows that allow light to pass through areas where a specific nucleotide is needed.
EXAMPLE:
If the mask is designed for the addition of thymine to the probe, you will find a window at that location, while probe locations where another base is required are blocked. Such a mask is laid on top of the array and ultraviolet light is shone onto it.
Probe locations that have windows are exposed to the light and the blocking compound removed from that probe, while covered locations still have the blocking compound present.
The quartz wafer is then washed with a solution of the desired nucleotide (e.g. thymine) that is linked to the same blocking compound, causing the nucleotide to attach to the probes that were exposed to light, while the nucleotide-attached blocking compound ensures that all of the probes are protected again. A capping step is added so that probes that did not attach their appropriate nucleotide are not incorrectly synthesized. The array is now ready for a new mask and the addition of a new nucleotide (cytosine in the figure). The process is repeated until all of the probes are complete (25 nucleotides each). Computer algorithms can calculate the optimal design of the masks that will minimize the number of reactions needed.
Once constructed, a microarray is ready for use in the laboratory.
WORKING OF MICROARRAYS
1. ISOLATION OF RNA FROM BIOLOGICAL SAMPLES
For microarray analysis, RNA can be isolated from a diverse set of biological samples using a combined phenol and glass-filter purification protocol. Each sample will contain thousands of different mRNA sequences representing all of the genes expressed in those cells.
2. GENERATION OF LABELED TARGETS:
Microarrays require 10-30 micrograms of labeled target per hybridization. Either labeled cDNA or aRNA (antisense) can be used for microarray experiments. In the Affymetrix system, the dNTPs are biotinylated, and later detection is performed with fluorescent streptavidin staining. In the simplest case, reverse transcription is performed using fluorescently tagged (e.g., Cy3 or Cy5), dinucleotide triphosphates (dNTPs), resulting in the generation of fluorescent cDNA (cDNA arrays).
2. TARGET HYBRIDIZATION.
The fluorescently labeled “targets,” are hybridized to gene-specific “probes.” Each target anneals to its corresponding probe spot on the microarray. The probes can be spotted cDNAs or oligonucleotides, or oligonucleotides that were synthesized directly on the micro-array surface. Stringent conditions are used to ensure that the probe sequences are entirely complementary to the micro-array spot sequences.
3. WASHING OF UNBOUND TARGETS
The slide is washed off to remove excess fluorescent targets not bound to spots.
4. DETECTION OF SIGNAL AND ANALYSIS OF DATA
After hybridization, a laser scanner is used to detect the specific fluorescence at each spot at two different wavelengths for green Cy3 or red Cy5. The colors of the spots are as follows:
Green = bound to Cy3-labeled cDNA, Red = bound to Cy5-labeled cDNA and Yellow = bound to both Cy3 and Cy5-labeled cDNA (these represent genes such as “housekeeping genes” that are required by all cells)
Software associated with the scanner allows identification of individual spots and measures each spot intensity. If all goes well, fluorescence intensity is proportional to the concentration of the relevant mRNA in the original sample.
DIAGRAMATIC REPRESENTATION OF MICROARRAY EXPERIMENT
The microarray is scanned with a laser beam, first at one wavelength to collect fluorescence data representing one probe, and then is scanned at a second wavelength to collect data representing the second probe. A computer compares the amount of fluorescence at each spot on the microarray for each probe. Through the use of computer software, the ratio of fluorescence is obtained and correlated with the clone address so the investigator knows which gene (spot on the slide) was expressed more in treated tissue as compared to control tissue.
OTHER TYPES OF MICROARRAYS
Apart from DNA microarrays, protein microarrays and tissue microarrays are the types most widely used.
1. TISSUE MICROARRAYS
Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. Tissue microarrays (TMA) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
Tissue microarrays are different from DNA microarrays where each spot on an array represents a cloned cDNA or oligonucleotide that binds to the target sequence. With tissue microarrays, each array has patient specific histological samples from cancer infected tissues. The tissue microarray technique is best suited for screening one genetic marker or protein across thousands of samples where as DNA microarrays are best suited to study gene expression across thousands of genes.
2. PROTEIN MICROARRAYS
In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyze antibody–antigen, protein–protein, protein–nucleic-acid, protein–lipid and protein–small-molecule interactions, as well as enzyme–substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyze entire proteomes.
APPLICATIONS OF MICROARRAYS
Microarrays can be used for large number of applications where high-throughput is needed. The current scope of microarray applications includes sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, and drug discovery and expression analysis.
1. SEQUENCE ANALYSIS
One of the earliest applications for microarrays was sequencing by hybridization. It is possible to determine the sequence of a target from the target’s pattern of hybridization with an array of all possible combinations of an oligonucleotide of a particular length. Subsequently, many other sequence-based applications have been developed, including genotyping, detection of point mutations, single nucleotide polymorphisms, and insertions and deletions, as well as the detection, identification, and enumeration of microorganisms.
2. GENE EXPRESSION ANALYSIS: GENE DISCOVERY
One of the most important applications of DNA Microarrays is the monitoring of gene expression where the abundance of the mRNA produced is determined for each gene. Such differences in gene expression are accountable for morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and perturbations. Changes in the multi-gene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways. In the context of medicine and treatment, such changes in patterns of expression can help to determine the causes and consequences of disease, how drugs and drug candidates work in cells and organisms.
Microarray technology has revolutionized studies of gene expression. A cDNA or oligonucleotide array complementary to expressed mRNA provides a means to simultaneously assess the expression of thousands of genes. Two-color labeling strategies introduce an additional degree of sophistication because they facilitate simultaneous comparison between gene expression in control and test cells on the same array. A goal in expression analysis is to place all of the genes for an organism on a single chip and then use that microarray to monitor changes in gene expression in cells. Already, there are chips with arrays representing all or many of the genes of the human and other species, such as rat, mouse, dog, monkey, honey bee, Arabidopis, Drosophila, and Escherichia coli.
3. DISEASE DIAGNOSIS:
DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.
4. DRUG DISCOVERY:
Drug discovery has evolved to become a large-scale and highly sophisticated endeavor. Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
5. TOXICOLOGICAL RESEARCH:
Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). The simultaneous measurement of the entire human genome (thousands of genes) will facilitate the uncovering of specific gene expression patterns that are associated with disease.
6. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) AND GENE COPY NUMBER
CGH is used as a molecular cytogenetic technique that permits quantitative analysis of gains and losses of whole or portions of chromosomes. A comprehensive high-resolution test for small chromosomal imbalances (microduplications or microdeletions) can now be performed in a single test. Investigators have developed a newer method that combines the principles of CGH with the use of microarrays. Instead of using metaphase chromosomes, this method—which is known as array CGH, or simply aCGH—uses slides arrayed with small segments of DNA as the targets for analysis. Microarray CGH works by comparing the levels of patient DNA to levels of a control (normal) DNA sample.In traditional CGH, DNAs from a "test" and a "reference" genome are differentially labeled with fluorophores and hybridized to normal metaphase chromosomes. In array CGH arrays of genomic BAC, P1, cosmid or cDNA clones are used for hybridization. CGH can not detect genomic changes involving <10–20 megabases. The use of microarray CGH overcomes many of these limitations, with improvement in resolution and dynamic range and improved throughput.
ROLE OF BIOINFORMATICS IN MICROARRAY TECHNOLOGY
Bioinformatics allows the researchers to tackle tasks easily by using Internet based tools without having to consider technical problems like local installation, maintenance or financial aspects. It aids in analysis of microarray technology by analyzing the expression levels, detecting anomalies, detecting SNP’s, finding GO terms, clustering, classification and then generating trees and processing fluorescent images.
TOOLS
1. Cryoscopy: Viewing a CGH (Comparative genomic hybridization) or expression data in a whole-genome context i.e. analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA. CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions cannot be detected, as they do not change the copy number.
2. Array Viewer: Identification of statistically significant hybridization signals.
3. Cluster: Perform hierarchical clustering and self-organizing maps.
4. BAM array: Bayesian Analysis of Variance for Microarrays detect differentially expressed genes from multigroup microarray data.
5. Expression Profiler: Analysis & clustering of gene expression data.
6. TreeView: Graphically browse results of clustering and other analyses from Cluster.
ROOM FOR IMPROVEMENT
AND
CONCLUSION
ROOM FOR IMPROVEMENT:
A lot of efforts have been made in the industry to improve the construction of the microarrays, as to advance the precision of the detection signals, increased speed of hybridization reactions, less usage of starting material and the cheaper availability of this technology.
"Microarrays will get better over time and a lot of that will be in content as we better understand which genes are important and, specifically, perhaps which splice variants are most important”, Says one of the scientists.
One of the improvements in the development phase is the usage of reflective substrates. It is speculated that printing microarrays on mirrors rather than glass improves the signal-to-noise ratio by as much as 1,000%.
CONCLUSION:
In recent decades, high-throughput scientific methods have been developed to optimize the study of large numbers of molecules, including DNA, proteins and metabolites. Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. DNA microarrays in particular have proved valuable in genomic research. They have been used to study gene expression patterns, to locate transcription factor binding sites, and to detect sequence mutations and deletions on a grand scale. However, DNA microarrays tell us only about the genes themselves and provide little information regarding the functions of the proteins they encode. Thus, protein microarrays were developed and were seen as high throughput approaches for the study of protein. On the other hand, tissue microarray is seen to provide a remarkable degree of standardization, speed, and cost efficiency for cancer analysis.
REFERENCES:
http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/basicbio.htm
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
http://www.chipscreen.com/articleFile/Microarrays-Tech%20and%20Application1.pdf
http://www.nature.com/nature/journal/v416/n6883/full/416885a.html
http://php.med.unsw.edu.au/cellbiology/index.php?title=Group_8_Project_-_Microarray
http://www.premierbiosoft.com/tech_notes/tissue-microarray.html
http://en.wikipedia.org/wiki/DNA_microarray
http://www.clinchem.org/cgi/content/full/47/8/1479
http://www.premierbiosoft.com/tech_notes/microarray.html
http://www.ncbi.nlm.nih.gov/geo/info/GEOHandoutFinal.pdf
http://www.sydneygenetics.com/Resources/WhatismicroarrayCGH/tabid/714/Default.aspx
http://www.ncbi.nlm.nih.gov/pubmed/12782098
http://chagall.med.cornell.edu/I2MT/MA-tools.pdf
http://discover.nci.nih.gov/tools.jsp
http://smd.stanford.edu/resources/restech.shtml
http://www.scq.ubc.ca/spot-your-genes-an-overview-of-the-microarray/
http://www.ambion.com/techlib/resources/microarray/basics1.html
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.fastol.com/~renkwitz/microarray_chips.htm
A cell functions by using its genes to produce proteins and although each cell within an organism will usually contain the same set of genes, there are significant differences in which genes are activated and how they are controlled because a fraction of these genes are turned on and it is the subset that is "expressed" that confers unique properties to each cell type. "Gene expression" is the term used to describe the transcription of the information contained within the DNA. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. For many years, study of a gene in this manner had to be done individually—looking at whether a specific gene is turned on (upregulated) or turned off (downregulated) under certain conditions. MICROARRAY is a technological advancement that has allowed scientists to study many, if not all, the genes of an organism’s at once. This high throughput method allows for the global study of changes in gene expression, giving us a complete cellular snapshot.
INTRODUCTION
Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. It is a high throughput technology that allows simultaneous detection of thousands of genes.
In formal terms a microarray can be defined as:
A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in a fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides. Microarrays are also referred to as DNA arrays, DNA chips, biochips and GeneChips.
Microarrays come up with the advantageous features as:
– Multiplexing: handling multiple samples at a time
– Parallelism: Studying Thousands of genes simultaneously
– Miniaturization: small sized chip
PRINCIPLE:
The principle of microarrays is based upon base paired hybridization probing, a technique that uses fluorescently labeled nucleic acid molecules as "mobile probes" to identify complementary molecules, and sequences that are able to base-pair with one another.
PARAMETERS DETERMINING THE NATURE OF A MICROARRAY TECHNOLOGY
PARAMETER OPTION
Probes: features arrayed on the microarray substrate that have known identity or sequence cDNA, oligonucleotides, proteins, peptide nucleic acids, small molecules, cells, tissues, and organisms
Fabrication: techniques to array probes on the microarray substrate In situ synthesis, robotic deposition
Targets: samples to be analyzed against the probes DNA, mRNA, proteins, enzymes, small molecules
Assays: principles based on which the targets are being analyzed Hybridization, electrophoresis, flow cytometry, ELISA
Signal readout: principles based on which the assay results can be detected Fluorescence , chemiluminescence, mass spectrometry, radioactivity, electrochemistry
Image processing: signal intensities of hybridized array spots are quantified from the scanning image Software for image processing
Informatics: computational tools with which the huge amount of data generated from a microarray experiment can be effectively stored and interpreted Database management system, data mining and visualization, interpretation of biological meaning
There are various types of microarrays, and to name a few, they include: DNA Microarrays, Protein Microarrays, Tissue Microarrays, Cellular Microarrays and Antibody Microarrays. Among the various types, DNA microarrays, protein microarrays and tissue microarrays are most widely used. The focus in the following sections will be on DNA microarrays.
DNA MICROARRAYS
DNA Microarray is the most widely used microarray technology. It is a type of nucleic acid-based multiplex technique involving high-density arrays of nucleic acids on glass that allows evaluating mRNA abundance of up to tens of thousands of genes simultaneously.
A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted (less than 1 mm) to that location. Through the use of highly accurate robotic spotters, over 30,000 spots can be placed on one slide, allowing molecular biologists to analyze virtually every gene present in a genome. The main advantage of microarrays is that the spots are single stranded DNA fragments that are strongly attached to the slide, allowing cellular DNA or RNA to be fluorescently labeled and laid overtop of the array. DNA or RNA in the overlaid sample will stick (through hybridization) to a complementary spot on the array, that is, Gene-A will stick to a spot composed of a Gene-A fragment. By exposing the microarray to a fluorescently labeled sample the DNA that hybridizes will be identifiable as glowing spots on the array, while the spots that have nothing hybridized to them will not be visible.
TYPES OF DNA MICROARRAYS
Two main types of commercial DNA microarrays are: oligonucleotide arrays and cDNA arrays.
1. OLIGONUCLEOTIDE ARRAYS
Oligonucleotide arrays (trademarked as a GeneChip by Affymetrix) use small 25 base pair gene fragments as the DNA probes to be spotted onto an array. These oligonucleotide probes are synthesized either in situ (on-chip) using photolithography or by conventional synthesis followed by on-chip immobilization.
APPLICATIONS OF OLIGONUCLEOTIDE ARRAYS
Oligonucelotide arrays are used for quantification of the amount of mRNA in a single sample (e.g. to determine the amount of mutated vs. non-mutated mRNA) or the comparison of two different samples hybridized to two separate arrays.
OLIGONUCLEOTIDE ARRAY TARGETS
Most array experimenters do not have access to micrograms of total RNA from their biological sample. A linear amplification strategy is highly recommended to obtain sample to be used for hybridization (targets). This technique consists of the following steps:
• First strand cDNA synthesis using reverse transcriptases;
• Generation of second strand cDNA using DNA polymerases; and
• In vitro transcription of the second strand cDNA to generate antisense RNA (aRNA) or complementary RNA (cRNA).
The probes are selected to have little cross-reactivity with other genes (targets) so that non-specific hybridization will be minimized.
2. cDNA ARRAYS
A cDNA array (also known as DNA microarray) is a different technology using the same principle; the probes in this case are larger pieces of DNA that are complementary to the genes one is interested in studying. With the cDNA probes prepared they can be mechanically spotted (robot spotting) onto a glass slide, normally in duplicate to serve as controls.
cDNA PROBES
• cDNA probes for making the array can also be generated from a commercially available cDNA library ensuring a close representation of the entire genome of an organism on the array.
• In an alternate target synthesis protocol, the target is generated by reverse transcription of the total RNA or the aRNA.
• Alternatively, PCR using specific primers can be used to amplify specific genes from genomic DNA to generate the cDNA probes.
When using cDNA spotted microarrays, two pools of RNA can be compared on the same array simultaneously. This is unlike Affymetrix GeneChip arrays, where RNA isolated from a single source is hybridized to an array or multiple arrays are required for comparison between more than one RNA samples.
DESIGN AND WORKING OF MICROARRAYS
DESIGN OF MICROARRAYS
Micro-array probes are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization.
• Micro-arrays are printed in situ (on chip) using a combination of photolithography and combinatorial chemistry.
• For on-chip immobilization, probes can be mechanically spotted onto a glass slide by using robot.
PHOTOLITHOGRAPHY:
The production begins with a 5-inch square of quartz wafer.
This wafer is washed with a blocking compound that can be removed by exposure to light. What is needed next is referred to as a ‘mask’, which is designed with 18-20 micron square windows that allow light to pass through areas where a specific nucleotide is needed.
EXAMPLE:
If the mask is designed for the addition of thymine to the probe, you will find a window at that location, while probe locations where another base is required are blocked. Such a mask is laid on top of the array and ultraviolet light is shone onto it.
Probe locations that have windows are exposed to the light and the blocking compound removed from that probe, while covered locations still have the blocking compound present.
The quartz wafer is then washed with a solution of the desired nucleotide (e.g. thymine) that is linked to the same blocking compound, causing the nucleotide to attach to the probes that were exposed to light, while the nucleotide-attached blocking compound ensures that all of the probes are protected again. A capping step is added so that probes that did not attach their appropriate nucleotide are not incorrectly synthesized. The array is now ready for a new mask and the addition of a new nucleotide (cytosine in the figure). The process is repeated until all of the probes are complete (25 nucleotides each). Computer algorithms can calculate the optimal design of the masks that will minimize the number of reactions needed.
Once constructed, a microarray is ready for use in the laboratory.
WORKING OF MICROARRAYS
1. ISOLATION OF RNA FROM BIOLOGICAL SAMPLES
For microarray analysis, RNA can be isolated from a diverse set of biological samples using a combined phenol and glass-filter purification protocol. Each sample will contain thousands of different mRNA sequences representing all of the genes expressed in those cells.
2. GENERATION OF LABELED TARGETS:
Microarrays require 10-30 micrograms of labeled target per hybridization. Either labeled cDNA or aRNA (antisense) can be used for microarray experiments. In the Affymetrix system, the dNTPs are biotinylated, and later detection is performed with fluorescent streptavidin staining. In the simplest case, reverse transcription is performed using fluorescently tagged (e.g., Cy3 or Cy5), dinucleotide triphosphates (dNTPs), resulting in the generation of fluorescent cDNA (cDNA arrays).
2. TARGET HYBRIDIZATION.
The fluorescently labeled “targets,” are hybridized to gene-specific “probes.” Each target anneals to its corresponding probe spot on the microarray. The probes can be spotted cDNAs or oligonucleotides, or oligonucleotides that were synthesized directly on the micro-array surface. Stringent conditions are used to ensure that the probe sequences are entirely complementary to the micro-array spot sequences.
3. WASHING OF UNBOUND TARGETS
The slide is washed off to remove excess fluorescent targets not bound to spots.
4. DETECTION OF SIGNAL AND ANALYSIS OF DATA
After hybridization, a laser scanner is used to detect the specific fluorescence at each spot at two different wavelengths for green Cy3 or red Cy5. The colors of the spots are as follows:
Green = bound to Cy3-labeled cDNA, Red = bound to Cy5-labeled cDNA and Yellow = bound to both Cy3 and Cy5-labeled cDNA (these represent genes such as “housekeeping genes” that are required by all cells)
Software associated with the scanner allows identification of individual spots and measures each spot intensity. If all goes well, fluorescence intensity is proportional to the concentration of the relevant mRNA in the original sample.
DIAGRAMATIC REPRESENTATION OF MICROARRAY EXPERIMENT
The microarray is scanned with a laser beam, first at one wavelength to collect fluorescence data representing one probe, and then is scanned at a second wavelength to collect data representing the second probe. A computer compares the amount of fluorescence at each spot on the microarray for each probe. Through the use of computer software, the ratio of fluorescence is obtained and correlated with the clone address so the investigator knows which gene (spot on the slide) was expressed more in treated tissue as compared to control tissue.
OTHER TYPES OF MICROARRAYS
Apart from DNA microarrays, protein microarrays and tissue microarrays are the types most widely used.
1. TISSUE MICROARRAYS
Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. Tissue microarrays (TMA) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.
Tissue microarrays are different from DNA microarrays where each spot on an array represents a cloned cDNA or oligonucleotide that binds to the target sequence. With tissue microarrays, each array has patient specific histological samples from cancer infected tissues. The tissue microarray technique is best suited for screening one genetic marker or protein across thousands of samples where as DNA microarrays are best suited to study gene expression across thousands of genes.
2. PROTEIN MICROARRAYS
In the past few years, protein microarray technology has shown its great potential in basic research, diagnostics and drug discovery. It has been applied to analyze antibody–antigen, protein–protein, protein–nucleic-acid, protein–lipid and protein–small-molecule interactions, as well as enzyme–substrate interactions. Recent progress in the field of protein chips includes surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production, and applications to analyze entire proteomes.
APPLICATIONS OF MICROARRAYS
Microarrays can be used for large number of applications where high-throughput is needed. The current scope of microarray applications includes sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, and drug discovery and expression analysis.
1. SEQUENCE ANALYSIS
One of the earliest applications for microarrays was sequencing by hybridization. It is possible to determine the sequence of a target from the target’s pattern of hybridization with an array of all possible combinations of an oligonucleotide of a particular length. Subsequently, many other sequence-based applications have been developed, including genotyping, detection of point mutations, single nucleotide polymorphisms, and insertions and deletions, as well as the detection, identification, and enumeration of microorganisms.
2. GENE EXPRESSION ANALYSIS: GENE DISCOVERY
One of the most important applications of DNA Microarrays is the monitoring of gene expression where the abundance of the mRNA produced is determined for each gene. Such differences in gene expression are accountable for morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and perturbations. Changes in the multi-gene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways. In the context of medicine and treatment, such changes in patterns of expression can help to determine the causes and consequences of disease, how drugs and drug candidates work in cells and organisms.
Microarray technology has revolutionized studies of gene expression. A cDNA or oligonucleotide array complementary to expressed mRNA provides a means to simultaneously assess the expression of thousands of genes. Two-color labeling strategies introduce an additional degree of sophistication because they facilitate simultaneous comparison between gene expression in control and test cells on the same array. A goal in expression analysis is to place all of the genes for an organism on a single chip and then use that microarray to monitor changes in gene expression in cells. Already, there are chips with arrays representing all or many of the genes of the human and other species, such as rat, mouse, dog, monkey, honey bee, Arabidopis, Drosophila, and Escherichia coli.
3. DISEASE DIAGNOSIS:
DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.
4. DRUG DISCOVERY:
Drug discovery has evolved to become a large-scale and highly sophisticated endeavor. Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.
5. TOXICOLOGICAL RESEARCH:
Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). The simultaneous measurement of the entire human genome (thousands of genes) will facilitate the uncovering of specific gene expression patterns that are associated with disease.
6. COMPARATIVE GENOMIC HYBRIDIZATION (CGH) AND GENE COPY NUMBER
CGH is used as a molecular cytogenetic technique that permits quantitative analysis of gains and losses of whole or portions of chromosomes. A comprehensive high-resolution test for small chromosomal imbalances (microduplications or microdeletions) can now be performed in a single test. Investigators have developed a newer method that combines the principles of CGH with the use of microarrays. Instead of using metaphase chromosomes, this method—which is known as array CGH, or simply aCGH—uses slides arrayed with small segments of DNA as the targets for analysis. Microarray CGH works by comparing the levels of patient DNA to levels of a control (normal) DNA sample.In traditional CGH, DNAs from a "test" and a "reference" genome are differentially labeled with fluorophores and hybridized to normal metaphase chromosomes. In array CGH arrays of genomic BAC, P1, cosmid or cDNA clones are used for hybridization. CGH can not detect genomic changes involving <10–20 megabases. The use of microarray CGH overcomes many of these limitations, with improvement in resolution and dynamic range and improved throughput.
ROLE OF BIOINFORMATICS IN MICROARRAY TECHNOLOGY
Bioinformatics allows the researchers to tackle tasks easily by using Internet based tools without having to consider technical problems like local installation, maintenance or financial aspects. It aids in analysis of microarray technology by analyzing the expression levels, detecting anomalies, detecting SNP’s, finding GO terms, clustering, classification and then generating trees and processing fluorescent images.
TOOLS
1. Cryoscopy: Viewing a CGH (Comparative genomic hybridization) or expression data in a whole-genome context i.e. analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA. CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions cannot be detected, as they do not change the copy number.
2. Array Viewer: Identification of statistically significant hybridization signals.
3. Cluster: Perform hierarchical clustering and self-organizing maps.
4. BAM array: Bayesian Analysis of Variance for Microarrays detect differentially expressed genes from multigroup microarray data.
5. Expression Profiler: Analysis & clustering of gene expression data.
6. TreeView: Graphically browse results of clustering and other analyses from Cluster.
ROOM FOR IMPROVEMENT
AND
CONCLUSION
ROOM FOR IMPROVEMENT:
A lot of efforts have been made in the industry to improve the construction of the microarrays, as to advance the precision of the detection signals, increased speed of hybridization reactions, less usage of starting material and the cheaper availability of this technology.
"Microarrays will get better over time and a lot of that will be in content as we better understand which genes are important and, specifically, perhaps which splice variants are most important”, Says one of the scientists.
One of the improvements in the development phase is the usage of reflective substrates. It is speculated that printing microarrays on mirrors rather than glass improves the signal-to-noise ratio by as much as 1,000%.
CONCLUSION:
In recent decades, high-throughput scientific methods have been developed to optimize the study of large numbers of molecules, including DNA, proteins and metabolites. Microarray technology has become a crucial tool for large-scale and high-throughput biology. It allows fast, easy and parallel detection of thousands of addressable elements in a single experiment. DNA microarrays in particular have proved valuable in genomic research. They have been used to study gene expression patterns, to locate transcription factor binding sites, and to detect sequence mutations and deletions on a grand scale. However, DNA microarrays tell us only about the genes themselves and provide little information regarding the functions of the proteins they encode. Thus, protein microarrays were developed and were seen as high throughput approaches for the study of protein. On the other hand, tissue microarray is seen to provide a remarkable degree of standardization, speed, and cost efficiency for cancer analysis.
REFERENCES:
http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/basicbio.htm
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
http://www.chipscreen.com/articleFile/Microarrays-Tech%20and%20Application1.pdf
http://www.nature.com/nature/journal/v416/n6883/full/416885a.html
http://php.med.unsw.edu.au/cellbiology/index.php?title=Group_8_Project_-_Microarray
http://www.premierbiosoft.com/tech_notes/tissue-microarray.html
http://en.wikipedia.org/wiki/DNA_microarray
http://www.clinchem.org/cgi/content/full/47/8/1479
http://www.premierbiosoft.com/tech_notes/microarray.html
http://www.ncbi.nlm.nih.gov/geo/info/GEOHandoutFinal.pdf
http://www.sydneygenetics.com/Resources/WhatismicroarrayCGH/tabid/714/Default.aspx
http://www.ncbi.nlm.nih.gov/pubmed/12782098
http://chagall.med.cornell.edu/I2MT/MA-tools.pdf
http://discover.nci.nih.gov/tools.jsp
http://smd.stanford.edu/resources/restech.shtml
http://www.scq.ubc.ca/spot-your-genes-an-overview-of-the-microarray/
http://www.ambion.com/techlib/resources/microarray/basics1.html
http://static.msi.umn.edu/tutorial/lifescience/IntroMicroarray.pdf
http://www.fastol.com/~renkwitz/microarray_chips.htm
Project on Morpholino
1. Background:
By 1984 a growing concern in the then-emerging antisense field was that antisense therapeutics might never be commercially viable because of their very high production costs.
Two key factors in the high cost of DNA analogs are:
1) The limited availability and high cost of their deoxyribonucleoside precursors;
2) The complexity and expense associated with coupling to hydroxyls, required in forming the phosphoester intersubunit linkages of most nucleic acid analogs.
In 1985 Summerton devised the Morpholino structural type to circumvent both of these cost problems. This is achieved by starting with much less expensive ribonucleosides and introducing an amine.
1. Introduction:
In molecular biology, a Morpholino is a molecule used to modify gene expression.
Morpholino oligonucleotides (oligos) are an antisense technology used to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid.
Morpholinos are usually used as a research tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein or by modifying the splicing of pre-mRNA. Knocking down gene expression is a powerful method for learning about the function of a particular protein; similarly, causing a specific exon to be spliced out of a protein can help to determine the function of the protein moiety encoded by that exon. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs, and sea urchins.
Morpholinos are also in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and for amelioration of genetic diseases. These synthetic oligos were conceived by James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dwight D. Weller ( AVI BioPharma Inc. )
2. Structure:
Morpholinos are synthetic molecules which 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. Structurally, the difference between Morpholinos and DNA is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
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 are most commonly used as single-stranded oligos, though heteroduplexes of a Morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagents.
4. Why Choose Morpholinos?
There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility, low toxicity and exquisite specificity. Only Morpholino oligos provide all of these.
Morpholinos provide much better specificity than RNAi, siRNA and phosphorothioate based oligos, greatly decreasing the chance of catastrophic off-target antisense effects.
The exquisite specificity of Morpholinos contributes to their extremely low toxicity and suitability for use in embryos, while off-target gene modulation makes siRNA unusable in most embryonic systems.
The non-ionic backbone of a Morpholino minimizes interactions with proteins, eliminating this mechanism for inducing non-antisense effects. Morpholinos do not trigger cellular responses by binding to Toll-like receptors, eliminating a confounding artifact associated with knockdowns using siRNA.
As steric blocking oligos, Morpholinos can be used not only to block translation but also to alter mRNA splicing, to bind miRNAs or to block binding of miRNA or regulatory proteins to RNA targets. Splice-blocking Morpholinos allow researchers to delete targeted exons and even analyze specific splice-forms of a gene with multiple splice variants. Morpholinos can target a pri-miRNA or a pre-miRNA to inhibit miRNA maturation or target a miRNA or one of its targets to inhibit miRNA activity. Binding of a splice-regulatory protein can be prevented by protecting its binding site with a Morpholino oligo.
5. Properties:
i. Solubility
An antisense oligo should have good water solubility to assure good access to its target sequence within the cell. Conventional perception in the antisense field is that non-ionic antisense oligos (such as Morpholinos) invariably show poor water solubility. Contrary to this perception, it has been found that if the nucleobases in a non-ionic antisense oligo stack poorly then that oligo has poor water solubility, but if the nucleobases are well stacked in aqueous solution then that oligo can show excellent water solubility.
It is assumed that the low water solubility of non-ionic oligos with poorly stacked bases is a result of the difficulty of inserting the hydrophobic faces of the unstacked bases into an aqueous environment. On the other hand, the high water solubility of non-ionic phosphorodiamidate-linked Morpholinos is likely due to effective shielding of those hydrophobic faces from the polar solvent because of the exceptionally good stacking of the bases.
ii. Stability
For optimal activity, an antisense oligo should be completely resistant to nucleases. DNA antisense oligos are degraded in serum and within cells in a matter of minutes.
Morpholino oligos are completely resistant to nucleases, as well as being resistant to a broad range of other degradative factors in biological systems - even including a liver homogenate. Accordingly, Morpholinos are effective in even long-term experiments and they are free of complications, which could arise from toxic degradation products.
iii. Efficiency: Cell-Free
It has been found that in a cell-free translation system with added RNase H the "old" 25-mer Morpholinos containing uracils typically exhibited slightly higher efficacies than corresponding S-DNAs, while "new" thymine-containing 25-mer Morpholinos generally achieve substantially higher efficacies than corresponding S-DNAs.
iv. Efficiency: In Cells
In experiments with Morpholinos carried out at ANTIVIRALS Inc. it has been found that Morpholino antisense oligos, which exhibit good activity in a cell-free translation system also, exhibit correspondingly good activity when scrape-loaded into cultured animal cells.
v. Binding Affinity
Targeting Morpholino oligos is vastly simpler than targeting DNA-based antisense oligos. Morpholinos usually work on the first attempt if they are successfully delivered to the cytosol. DNA and S-DNA oligos have a low probability of shutting down a target gene on the first attempt; it is common for five or six oligos to be tried before an effective sequence is found.
This is in part because a Morpholino oligo has a higher binding affinity than an equivalent DNA-based antisense oligo. This higher affinity allows Morpholinos to invade RNA secondary structure and substantially increases the probability of designing effective oligos. Precisely targeting a single gene is also simplified because Morpholino oligos act via steric-blocking as opposed to an RNAse H-mediated mRNA degradation mechanism.
6. Function of Morpholinos
Morpholinos do not degrade their target RNA molecules, unlike many antisens structural types (e.g. siRNA). 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.
Normal gene expression in eukaryotes
Eukaryotic gene expression without intervention by a Morpholinos
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 to one end of the mRNA and 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 a sequence of bases in DNA, is converted into the structure of a protein. A morpholino can modify splicing or block translation, depending on the morpholino's base sequence.
i. Blocking translation
Translation blocked by a Morpholino oligo
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 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.
ii. Modifying pre-mRNA splicing
Splicing blocked by a Morpholino oligo
Morpholinos can interfere with pre-mRNA processing steps by:
• preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA
• by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure
• by interfering with the binding of splice regulatory proteins such as splice silencer and splice enhancers.
Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction.
iii. Blocking other mRNA sites
Morpholinos have been used to block miRNA activity and maturation.They can also block ribozyme activity. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.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.
7. Applications of Morpholinos:
There are three main applications of morpholinos which are:
a) Blocking mRNA translation
b) Blocking nuclear processing
c) Anti-sense therapeutics
Let us explain them one by one.
a) Blocking translation:
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 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, the targeted proteins become undetectable by Western blot.
b) Blocking nuclear processing:
Morpholinooligos can block nuclear processing events, pre-mRNA processing in particular. The power of high specificity and steric blocking allows one to specifically and reproducibly delete exons of choice by blocking access of the splicing machinery to the pre-mRNA. This technology, not possible with RNase-dependent or RISCdependentoligos (phosphorothioates, RNAi and others), not only allows characterizing specific exon function and creating loss-of-function deletions or insertions but it also allows researchers to eliminate a specific splice variant while leaving anothersplice variant of the same gene intact.
c) Anti-sense therapeutics:
Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process,
such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs.
8. DELIVERY OF MORPHOLINOS
To make a morpholino effective we have to effectively deliver it to the cytosol through the cell membrane. In cytosol they can freely diffuse and start functioning. Different methods are used for delivering morpholinos, some of which are mentioned below:
i. Delivery to Embryo:
Microinjection:
A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage.
Electroporation:
An alternative method for embryonic delivery is electroporation, which can deliver oligos into tissues of later embryonic stages.
“Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.”
ii. Delivery to Cultured cells:
One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems.
So as a solution a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electro statically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol.
iii. Delivery to Adult cells:
Use of cell-penetrating peptides to help Morpholinos into cells, primarily through an endocytotic pathway followed by release from the endosome.
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.
9. Vivo morpholinos
Vivo-Morpholinos are the knockdown, exon-skipping or miRNA blocking reagent of choice for in vivo experiments. Outstanding results can be achieved systemically with intravenous injection, and modest systemic delivery achieved with Vivo-Morpholinos by intraperitoneal (I.P.) injection. Efficient localized delivery can be achieved by injecting the Vivo-Morpholinos directly into the area of interest. If you wish to initially validate the oligo in cultured cells you can use the same Vivo-Morpholinos in cultures. However, unmodified Morpholinos, delivered with either electroporation or Endo-Porter, are more efficient in cell cultures than Vivo-Morpholinos
i. Composition:
A Morpholino molecule consists of a Morpholino oligo with a unique covalently linked delivery moiety, which is comprised of an octa-guanidine dendrimer. It uses the active component of arginine rich delivery peptides (the guanidinium group) with improved stability, low toxicity and reduced cost.
The Vivo-Morpholino is assembled by coupling the vivo-delivery group to a Morpholino while the oligo is still bound to its synthesis resin, allowing excellent purification by washing the solid-phase resin. Vivo-Morpholinos must be chosen prior to synthesis and cannot be added later because the vivo-delivery group is added to a Morpholino prior to cleavage from its synthesis resin.
ii. Delivery and dosage:
For best systemic delivery results the injection method of choice is I.V. although I.P. can also be used. The maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. This can be repeated daily. The dosage for short term and long term experiment differ. For short term the experiment lasts 2-3 days the dosage can be 12.5 mg/kg. While for the long term experiments the dosage is preferably started at dosage equivalent to the short term. The dosage and duration can be increased to optimize the results.
One can expect quantifiable knockdown or exon-skipping in liver, small intestine, colon, muscle, lung, and stomach tissues with lesser but quantifiable delivery in the spleen, heart, skin and brain.
It has been found that younger or older mice do not tolerate Vivo-Morpholinos as well and may require substantially lower doses. In addition the use of compromised mice, such as those with less robust genetic backgrounds, may require further dose limitations.
10. Problem Assosiated with Morpholinos:
Occasionally Morpholino oligos can fail and there are many factors which can contribute to oligo failure, some correctable ones determined.
1) Morpholino Oligos can lose activity if improperly stored or stored in anything other than sterile pure water. It needs to be stay fluffy and smooth, and should not turn hard palette like. To avoid this, if the oligo is to be stored long term keep it in a desiccator at room temperature. If the oligo was fluffy and there is still problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as lissamine fluorescent tag. In these cases, try heating to 65C for 10 minutes and vortexing. Leaving the oligo overnight on a vigorous shaker might also help dissolution and Continue until completely resuspended.
2) Stock Morpholino oligo should be dissolved in sterile water without Diethylpyrocarbonate (DEPC) because DEPC forms bond with morphiline ring in the oligos backbone. It is possible for an oligo to lose activity if they have undergone long-term exposure to acid or DEPC.
3) Some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration because of high expressivity.
4) Specificity is reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.
5) There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo. The secondary targets can come from homologous genes. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo since single-molecule stem-loops do not readily form due to the oligo's limited flexibility, but inter-strand pairing can tie-up oligo and thus eliminate it from the pool of oligo that can pair with target sequence.
6) We cannot target point mutations with morpholinos. The property which gives Morpholino oligos their spectacular sequence specificity also largely precludes their use for effectively targeting point mutations. However, Morpholinos are the best tools for studying point mutations, polymorphisms, and a host of other genetic variations using the gene switch strategy or splice-blocking.
7) we cannot target the amino acid coding region of mRNA with morpholinos, because Morpholino oligos functions solely by a steric blocking mechanism, only the leader sequence and the first 25 bases of the amino acid coding region can be targeted for translation blocking applications.
8) Morpholino oligos enter unperturbed tissue culture cells but high concentrations (tens of µM) and long incubation times (multiple days) are required to achieve even very modest amounts of delivery in most cell types.
9) The following cells can NOT be delivered with the Morpholinos
a. All T-cell derived lines (apparently do not endocytose)
b. All differentiated colon carcinoma cell lines (also apparently do not endocytose)
10) Non-ionic Morpholinos will not complex with cationic lipids for delivery
11. Alternates of morpholino:
i. Small interfering RNA (siRNA):
Also known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".
ii. MicroRNAs (miRNAs):
MicroRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger (mRNAs), usually resulting in translational repression and silencing. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian gene and are abundant in many human cell types.
Reference:
http://www.gene-tools.com/node/12
http://network.nature.com/groups/drugdelivery/forum/topics/359
http://nar.oxfordjournals.org/content/early/2007/08/01/nar.gkm478.full.pdf+html
http://www.reference.com/browse/morpholino
http://morpholinos.yuku.com/
http://www.gene-tools.com/node/25
http://www.gene-tools.com/faq
By 1984 a growing concern in the then-emerging antisense field was that antisense therapeutics might never be commercially viable because of their very high production costs.
Two key factors in the high cost of DNA analogs are:
1) The limited availability and high cost of their deoxyribonucleoside precursors;
2) The complexity and expense associated with coupling to hydroxyls, required in forming the phosphoester intersubunit linkages of most nucleic acid analogs.
In 1985 Summerton devised the Morpholino structural type to circumvent both of these cost problems. This is achieved by starting with much less expensive ribonucleosides and introducing an amine.
1. Introduction:
In molecular biology, a Morpholino is a molecule used to modify gene expression.
Morpholino oligonucleotides (oligos) are an antisense technology used to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid.
Morpholinos are usually used as a research tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein or by modifying the splicing of pre-mRNA. Knocking down gene expression is a powerful method for learning about the function of a particular protein; similarly, causing a specific exon to be spliced out of a protein can help to determine the function of the protein moiety encoded by that exon. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs, and sea urchins.
Morpholinos are also in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and for amelioration of genetic diseases. These synthetic oligos were conceived by James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dwight D. Weller ( AVI BioPharma Inc. )
2. Structure:
Morpholinos are synthetic molecules which 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. Structurally, the difference between Morpholinos and DNA is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
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 are most commonly used as single-stranded oligos, though heteroduplexes of a Morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagents.
4. Why Choose Morpholinos?
There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility, low toxicity and exquisite specificity. Only Morpholino oligos provide all of these.
Morpholinos provide much better specificity than RNAi, siRNA and phosphorothioate based oligos, greatly decreasing the chance of catastrophic off-target antisense effects.
The exquisite specificity of Morpholinos contributes to their extremely low toxicity and suitability for use in embryos, while off-target gene modulation makes siRNA unusable in most embryonic systems.
The non-ionic backbone of a Morpholino minimizes interactions with proteins, eliminating this mechanism for inducing non-antisense effects. Morpholinos do not trigger cellular responses by binding to Toll-like receptors, eliminating a confounding artifact associated with knockdowns using siRNA.
As steric blocking oligos, Morpholinos can be used not only to block translation but also to alter mRNA splicing, to bind miRNAs or to block binding of miRNA or regulatory proteins to RNA targets. Splice-blocking Morpholinos allow researchers to delete targeted exons and even analyze specific splice-forms of a gene with multiple splice variants. Morpholinos can target a pri-miRNA or a pre-miRNA to inhibit miRNA maturation or target a miRNA or one of its targets to inhibit miRNA activity. Binding of a splice-regulatory protein can be prevented by protecting its binding site with a Morpholino oligo.
5. Properties:
i. Solubility
An antisense oligo should have good water solubility to assure good access to its target sequence within the cell. Conventional perception in the antisense field is that non-ionic antisense oligos (such as Morpholinos) invariably show poor water solubility. Contrary to this perception, it has been found that if the nucleobases in a non-ionic antisense oligo stack poorly then that oligo has poor water solubility, but if the nucleobases are well stacked in aqueous solution then that oligo can show excellent water solubility.
It is assumed that the low water solubility of non-ionic oligos with poorly stacked bases is a result of the difficulty of inserting the hydrophobic faces of the unstacked bases into an aqueous environment. On the other hand, the high water solubility of non-ionic phosphorodiamidate-linked Morpholinos is likely due to effective shielding of those hydrophobic faces from the polar solvent because of the exceptionally good stacking of the bases.
ii. Stability
For optimal activity, an antisense oligo should be completely resistant to nucleases. DNA antisense oligos are degraded in serum and within cells in a matter of minutes.
Morpholino oligos are completely resistant to nucleases, as well as being resistant to a broad range of other degradative factors in biological systems - even including a liver homogenate. Accordingly, Morpholinos are effective in even long-term experiments and they are free of complications, which could arise from toxic degradation products.
iii. Efficiency: Cell-Free
It has been found that in a cell-free translation system with added RNase H the "old" 25-mer Morpholinos containing uracils typically exhibited slightly higher efficacies than corresponding S-DNAs, while "new" thymine-containing 25-mer Morpholinos generally achieve substantially higher efficacies than corresponding S-DNAs.
iv. Efficiency: In Cells
In experiments with Morpholinos carried out at ANTIVIRALS Inc. it has been found that Morpholino antisense oligos, which exhibit good activity in a cell-free translation system also, exhibit correspondingly good activity when scrape-loaded into cultured animal cells.
v. Binding Affinity
Targeting Morpholino oligos is vastly simpler than targeting DNA-based antisense oligos. Morpholinos usually work on the first attempt if they are successfully delivered to the cytosol. DNA and S-DNA oligos have a low probability of shutting down a target gene on the first attempt; it is common for five or six oligos to be tried before an effective sequence is found.
This is in part because a Morpholino oligo has a higher binding affinity than an equivalent DNA-based antisense oligo. This higher affinity allows Morpholinos to invade RNA secondary structure and substantially increases the probability of designing effective oligos. Precisely targeting a single gene is also simplified because Morpholino oligos act via steric-blocking as opposed to an RNAse H-mediated mRNA degradation mechanism.
6. Function of Morpholinos
Morpholinos do not degrade their target RNA molecules, unlike many antisens structural types (e.g. siRNA). 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.
Normal gene expression in eukaryotes
Eukaryotic gene expression without intervention by a Morpholinos
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 to one end of the mRNA and 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 a sequence of bases in DNA, is converted into the structure of a protein. A morpholino can modify splicing or block translation, depending on the morpholino's base sequence.
i. Blocking translation
Translation blocked by a Morpholino oligo
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 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.
ii. Modifying pre-mRNA splicing
Splicing blocked by a Morpholino oligo
Morpholinos can interfere with pre-mRNA processing steps by:
• preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA
• by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure
• by interfering with the binding of splice regulatory proteins such as splice silencer and splice enhancers.
Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction.
iii. Blocking other mRNA sites
Morpholinos have been used to block miRNA activity and maturation.They can also block ribozyme activity. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.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.
7. Applications of Morpholinos:
There are three main applications of morpholinos which are:
a) Blocking mRNA translation
b) Blocking nuclear processing
c) Anti-sense therapeutics
Let us explain them one by one.
a) Blocking translation:
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 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, the targeted proteins become undetectable by Western blot.
b) Blocking nuclear processing:
Morpholinooligos can block nuclear processing events, pre-mRNA processing in particular. The power of high specificity and steric blocking allows one to specifically and reproducibly delete exons of choice by blocking access of the splicing machinery to the pre-mRNA. This technology, not possible with RNase-dependent or RISCdependentoligos (phosphorothioates, RNAi and others), not only allows characterizing specific exon function and creating loss-of-function deletions or insertions but it also allows researchers to eliminate a specific splice variant while leaving anothersplice variant of the same gene intact.
c) Anti-sense therapeutics:
Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process,
such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs.
8. DELIVERY OF MORPHOLINOS
To make a morpholino effective we have to effectively deliver it to the cytosol through the cell membrane. In cytosol they can freely diffuse and start functioning. Different methods are used for delivering morpholinos, some of which are mentioned below:
i. Delivery to Embryo:
Microinjection:
A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage.
Electroporation:
An alternative method for embryonic delivery is electroporation, which can deliver oligos into tissues of later embryonic stages.
“Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.”
ii. Delivery to Cultured cells:
One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems.
So as a solution a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electro statically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol.
iii. Delivery to Adult cells:
Use of cell-penetrating peptides to help Morpholinos into cells, primarily through an endocytotic pathway followed by release from the endosome.
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.
9. Vivo morpholinos
Vivo-Morpholinos are the knockdown, exon-skipping or miRNA blocking reagent of choice for in vivo experiments. Outstanding results can be achieved systemically with intravenous injection, and modest systemic delivery achieved with Vivo-Morpholinos by intraperitoneal (I.P.) injection. Efficient localized delivery can be achieved by injecting the Vivo-Morpholinos directly into the area of interest. If you wish to initially validate the oligo in cultured cells you can use the same Vivo-Morpholinos in cultures. However, unmodified Morpholinos, delivered with either electroporation or Endo-Porter, are more efficient in cell cultures than Vivo-Morpholinos
i. Composition:
A Morpholino molecule consists of a Morpholino oligo with a unique covalently linked delivery moiety, which is comprised of an octa-guanidine dendrimer. It uses the active component of arginine rich delivery peptides (the guanidinium group) with improved stability, low toxicity and reduced cost.
The Vivo-Morpholino is assembled by coupling the vivo-delivery group to a Morpholino while the oligo is still bound to its synthesis resin, allowing excellent purification by washing the solid-phase resin. Vivo-Morpholinos must be chosen prior to synthesis and cannot be added later because the vivo-delivery group is added to a Morpholino prior to cleavage from its synthesis resin.
ii. Delivery and dosage:
For best systemic delivery results the injection method of choice is I.V. although I.P. can also be used. The maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. This can be repeated daily. The dosage for short term and long term experiment differ. For short term the experiment lasts 2-3 days the dosage can be 12.5 mg/kg. While for the long term experiments the dosage is preferably started at dosage equivalent to the short term. The dosage and duration can be increased to optimize the results.
One can expect quantifiable knockdown or exon-skipping in liver, small intestine, colon, muscle, lung, and stomach tissues with lesser but quantifiable delivery in the spleen, heart, skin and brain.
It has been found that younger or older mice do not tolerate Vivo-Morpholinos as well and may require substantially lower doses. In addition the use of compromised mice, such as those with less robust genetic backgrounds, may require further dose limitations.
10. Problem Assosiated with Morpholinos:
Occasionally Morpholino oligos can fail and there are many factors which can contribute to oligo failure, some correctable ones determined.
1) Morpholino Oligos can lose activity if improperly stored or stored in anything other than sterile pure water. It needs to be stay fluffy and smooth, and should not turn hard palette like. To avoid this, if the oligo is to be stored long term keep it in a desiccator at room temperature. If the oligo was fluffy and there is still problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as lissamine fluorescent tag. In these cases, try heating to 65C for 10 minutes and vortexing. Leaving the oligo overnight on a vigorous shaker might also help dissolution and Continue until completely resuspended.
2) Stock Morpholino oligo should be dissolved in sterile water without Diethylpyrocarbonate (DEPC) because DEPC forms bond with morphiline ring in the oligos backbone. It is possible for an oligo to lose activity if they have undergone long-term exposure to acid or DEPC.
3) Some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration because of high expressivity.
4) Specificity is reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.
5) There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo. The secondary targets can come from homologous genes. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo since single-molecule stem-loops do not readily form due to the oligo's limited flexibility, but inter-strand pairing can tie-up oligo and thus eliminate it from the pool of oligo that can pair with target sequence.
6) We cannot target point mutations with morpholinos. The property which gives Morpholino oligos their spectacular sequence specificity also largely precludes their use for effectively targeting point mutations. However, Morpholinos are the best tools for studying point mutations, polymorphisms, and a host of other genetic variations using the gene switch strategy or splice-blocking.
7) we cannot target the amino acid coding region of mRNA with morpholinos, because Morpholino oligos functions solely by a steric blocking mechanism, only the leader sequence and the first 25 bases of the amino acid coding region can be targeted for translation blocking applications.
8) Morpholino oligos enter unperturbed tissue culture cells but high concentrations (tens of µM) and long incubation times (multiple days) are required to achieve even very modest amounts of delivery in most cell types.
9) The following cells can NOT be delivered with the Morpholinos
a. All T-cell derived lines (apparently do not endocytose)
b. All differentiated colon carcinoma cell lines (also apparently do not endocytose)
10) Non-ionic Morpholinos will not complex with cationic lipids for delivery
11. Alternates of morpholino:
i. Small interfering RNA (siRNA):
Also known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".
ii. MicroRNAs (miRNAs):
MicroRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger (mRNAs), usually resulting in translational repression and silencing. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian gene and are abundant in many human cell types.
Reference:
http://www.gene-tools.com/node/12
http://network.nature.com/groups/drugdelivery/forum/topics/359
http://nar.oxfordjournals.org/content/early/2007/08/01/nar.gkm478.full.pdf+html
http://www.reference.com/browse/morpholino
http://morpholinos.yuku.com/
http://www.gene-tools.com/node/25
http://www.gene-tools.com/faq
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