Saturday, February 28, 2009

RNA Interference Complete Overview

This report on siRNA aims to sumarize the current state of knowledge of the pioneering work of Fire, Mello and colleagues at the University of Massachussets. The idea to control gene expression, perhaps go back to the early days of antisense approaches, whereby a single stranded oligonucleotide could be designed,complimentary to the corresponding mRNA in order to interfere with its translation. Likewise in those days, stability and penetration were also issues to contend with; both exo and endonucleases diminished the effect of the AS oligos; then various modifications were introduced to the diester backbone, including but not limited to sulfur , methyl, ethyl and other moieties, with the aim to extend the net half life of the AS oligos . Penetration wise, the AS oligos needed to be applied a high concentrations and the time of gene suppression was generally short lived; cationic based, transfection agents also were required. Amazingly most short RNA’s that most biological researchers dealt with, generally were indication of bad technique/degradation and were summarily discarded. Enter 1992, and the finding that indeed there was a natural gene inhibititon mechanism, whereby dsRNA was found to be used in Caenorhabditis elegans , now it is also known that RNAi control of gene expression is a general mechanism found also in plants and mamalian organisms. In this review we go over the known mechanism of Dicer splicing to produce 21-23 nts, with 2 base overhangs on both ends. This is followed by RISC interaction where the guide strand is prepared for final mRNA scission. The fine details occurring at RISC, where enzymes like Drosha, Pasha and others, interplay to finally produce the guide strand that then is ready to potently initiate mRNA cleavage, are still on the works. Chemical and in vitro approaches are also covered to give the reader the various alternatives to obtain inhibition of the gene of interest. Chemical modifications and delivery approaches are also discussed; it is noteworthy to mention the phoshonoacetate approach, where the ODN’s themselves possess a unique penetration ability in a wide variety of human cell lines. PDF document provide following inforamtion:

* RNAi in drug discovery and disease therapy
* RNAi for genetic diseases

Absolute Quantification of Specific Proteins in Complex Mixtures Using Visible Isotope-Coded

This article illustrate to us the use of a novel type of protein tagging reagent, that the authors call , “the visible isotope-coded affinity tag” (VICAT) which permits the quantification of the absolute amount of a target protein/s within a highly complex biological sample such as a eukaryotic cell lysate. This method can tag the thiol groups of cysteines or thioacetylated amino groups and also can introduce a biotin affinity handle (a visible moiety ) which allows tracking of the chromatographic position of the target peptide, without the need of a mass spectrometrer. A photocleavable linker (for tag removal) and an isotopic tag are also introduced, which enables distinguishing between the sample and reference peptides. The authors show the application of VICAT reagents to determine the absolute abundance of human group V phospholipase A2, in eukaryotic cell lysates;combining isolectric focusing of peptides on immobilized gel strip followed by microliquid chromatography/electrospray ionization mass spectrometry, they show that 66 fmol of phospholipase A2 per 100 ugs of cell protein are found in human lung macrophages. On the other hand Western blotting did not provide conclusive results. It is envisioned that VICAT reagents may be helpful for various applications including but not limited to the analysis of lead disease markers that could be detected in serum samples.

Friday, February 27, 2009

Silencing of microRNAs in vivo with ‘antagomirs’

To develop a pharmacological approach for silencing miRNAs in vivo, a small interfering double-stranded RNAs (siRNAs) engineerred with certain "drug-like" properties such as chemical modifications for stability and cholesterol conjugation for delivery have been shown to achieve therapeutic silencing fo an endogeous gene in vivo. To explore the potential of these synthetic RNA analogues to silence endogenous miRNAs, chemical modified, cholesterol-conjugated antagomir, antagomir-122 which sequence designed for miR-122, an abundant, liver-specific miRNA. Antagomir-122 was synthesized starting from a hydroxyprolinol-linked cholesterol solid support14 and 2''-OMe phosphoramidites. This compound was administered to mice by intravenous injection at normal pressure. Administration of antagomir-122 resulted in a marked decrease in endogenous miR-122 levels as detected by northern blots while unmodified single-stranded RNA (anti-122) had no effect on levels of hepatic miRNA-122. Click on PDF link below for this article!

A Novel Lipid Hydroperoxide-derived Cyclic Covalent Modification

In this paper we learn that that 4-hydroxy-2- nonenal (a lipid hydroperoxide-derived aldehydic bifunctional electrophile) reacts with DNA and proteins and furthermore the authors observe that 4-oxo- 2-nonenal is also a major product of lipid hydroperoxide decomposition. Moreover, 4-oxo-2-nonenal appears to be more reactive than 4-hydroxy-2-nonenal toward the DNAbases 2’-deoxyguanosine, 2’-deoxyadenosine, and 2’-deoxycytidine it is pointed out that homolytic decomposition of polyunsaturated lipid hydroperoxides such as 13(S)-hydroperoxyoctadecadienoic acid, can lead to formation of 4-oxo-2-nonenal. Very importantly the authors report that either synthetic 4-oxo-nonenal or 4-oxo-2-nonenal- generated from 13(S)-hydroperoxyoctadecadienoic can interact and recognize with His75, Ala76, and Lys77 of bovine histone H4. The amino acid residues histidine and lysine react with 4-oxo-2-nonenal to form a new cyclic structure within the protein. Such cyclic structures integrate the histidine imidazole ring with the pyrrole ring derived from lysine. This cyclic imidazole-pyrrole derivative formed from N-acetyl-His-Ala-Lys peptide can be found as a mixture of two atropisomers that interchange upon heating. It is conceived that this lipid hydroperoxide moeities may be able to modulate transcriptional activation in vivo. Novel chemical synthesis of structural analogs of these cyclic peptides could generate new compounds with improved biological activity.

Friday, February 20, 2009

Essential versus Non-Essential Amino Acids

The human body requires 20 naturally occurring amino acids for its proper functioning. There are 8 essential amino acids for humans: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, and lysine. They are called essential because the body does not manufacture them but must be ingested in the diet. Therefore a proper diet should be balanced and must include all the essential amino acids. Foods that contain a good and varied amount of amino acids, including the essential ones should be included in our daily diet. Plant sources such us quinoa, buckwheat, hempseed, amaranth, among others contain a balanced set of amino acids.

Peptides and Amino Acids

What is an amino acid? Is a molecule made up of carbon, hydrogen and nitrogen that make up peptides and proteins; there are 20 naturally occurring amino acids found in most living organisms

What is a peptide? Peptides are strings of amino acids that make up essential components of our bodies; proteins and enzymes are made up of various lengths of amino acids. The human body requires of 20 amino acids for its proper functioning.

The word peptides or oligopeptides refer to strings of amino acids(AA’s) of up to 50 AA’s (this is an arbitrary length); is used to primarily to differentiate between short and long strings of AA’s; long strings of AA’s are known as proteins; keep in mind that in many situations the words can be used interchangeably.

For example human insulin is a 51 amino acid long hormone/protein; it can be referred as a peptide or protein (in any case it is made up of a string of amino acids). Insulin is a peptide and has a molecular weight of 5808 Daltons. It is produced and secreted by the islets of Langerhans in the pancreas. Various deficiencies of insulin produce diabetes.

Bioconjugates: Development and Applications of Nucleic Acids and Peptides Conjugated to Lipids and/or Carbohydrates

The availability of synthetic nucleic acids/oligonucleotides and peptides, areas where BioSynthesis has extensive experience, has promoted the development of conjugates of these molecules cross-linked to compounds such as lipids, carbohydrates and small molecules (drugs) to yield products with distinct properties. Taking advantage of its expertise in nucleic acids and peptide synthesis, BioSynthesis is developing new approaches for the production of different bioconjugates. From the point of view of therapeutic applications, because of their potential uses in genetic therapy and blocking gene expression the bioconjugates of nucleic acids are the most important ones. In general these bioconjugates acquire new properties, such as resistance to degradation and passage of the hydrophilic and anionic oligonucleotide across the hydrophobic negatively charged cell membrane or transduction. Yet, a suitable bioconjugate should maintain practically all of the oligonucleotide’s biological properties.

Lipid-oligonucleotide conjugates The main objective of cross-linking a lipid moiety to an oligonucleotide, either an oligodeoxynucleotide (ODN) or oligoribonucleotide (ORN), is to increase the hydrophobic character of the latter and its lipid-solubility. This way a conjugate would pass across the highly lipophilic cell membrane and into the cytosol, a process called transduction. Yet, depending on the lipid’s nature these conjugates may have also some other new biological properties.

Cholesterol is a lipid that has been extensively used in the production of these conjugates. The cholesterol tag can be added at the 3’ or 5’ end of an ODN usually using a C4- to C8-linker or a polyethylene glycol linker. Cholesterol-ODN conjugates in addition to better transduction show improved nuclease resistance and anti-viral activity. In addition, these conjugates can form very stable duplex and triplex structures, with some small 3’-cholesterol modified ODN duplexes showing anti-tumor activity. Conjugation of antisense phosphorothioate ODN with cholesterol yields compounds with a higher antisense activity than their unconjugated counterparts. Cholesterol has been also used to produce conjugates of siRNA showing an improved cellular uptake. Because of its nature, siRNA when delivered as a complex with cationic lipids into the cell’s endosomes are recognized by TLR7 and TLR8, leading to the production of inflammatory cytokines. Replacement of the cationic lipids by covalently bound cholesterol does not stimulate such an immune response. Cholesterol can be replaced by plant-derived sterols, steroids and other related compounds (Fig 1). An advantage of using these products as tags is that frequently they target specific cell receptors, thus providing some degree of tissue specificity. Cholesterol and other natural and synthetic sterols can also be used to make conjugates of peptide nucleic acids (PNA).

In addition to cholesterol and other sterols, additional lipids such as alkyl chains, phospholipids, fatty acids and lipid substituted crown ethers can also be used (Fig 1). Like cholesterol-ODN conjugates, ODNs conjugated to alkyl chains larger than 12 carbons would bind to the serum lipoproteins LDL and HDL to form complexes that are taken up by these proteins’ cell receptors facilitating their entry. Lipophilic dendrimers have been conjugated to either the 5’- or 3’-ends of antisense ODNs to increase their cellular uptake. Increase in the size of the conjugate’s dendrimer results in a significant decrease in binding activity as shown by a marked drop in melting temperature.

Another approach to ODN delivery is the use of ODN conjugates of polyethylene glycol (PEG) that has been applied to antisense ODN. PEG-ODN conjugates have a diblock copolymer-like structure where the ODN segment can interact with a cationic fusogenic peptide (rich in lysine and arginine, KALA) to form a polyelectrolyte core while the PEG segments are protruding from the micelle. These micelles are taken up by the cells via endocytosis to effectively deliver the antisense ODNs to their endosomal compartment. The KALA peptide associated to the ODN by disrupting the endosomal membrane allows the antisense ODN to enter the cell’s cytosol. The conjugation process Cross-linking of lipids to oligonucleotides can usually take place at either their carbohydrate moiety, deoxyribose or ribose, or their single terminal 5’ phosphate group. Cholesterol with a spacer can be obtained by reacting the cholesteryl-chloroformate with 6-amino-1-hexanol to yield a C6 spacer linked to cholesterol by an amide bond. However, the length of the spacer can be from 3 to 8 carbons. The terminal –OH of this spacer can then be phosphitylated with 2-cyanoethyl-N,N,N’,N’ tetraisopropyl phosphoramidite to yield a phosphoramidite that would react with the 5’-OH of the terminal deoxyribose (Fig. 2).

In ORNs where the 2’ –OH from the riboses are protected, the intermediate would react with the unprotected 5’ –OH. The oligonucleotide will be cross-linked to cholesterol via a spacer (Fig. 2). Cholesterol can also be added to the terminal 5’ phosphate of an oligonucleotide by reacting the cholesteryl-chloroformate with a diamine’s excess, e.g. ethylenediamine, to yield an aminated cholesteryl that is separated from the excess of diamine using silica gel chromatography. The aminated cholesteryl upon reaction with succinic anhydride would yield a larger side chain with a terminal carboxyl group (Fig. 3-A) that can be activated with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The activated cholesteryl derivative is them reacted with an oligonucleotide that has an amino alkyl group linked to the 5’ phosphate (Fig. 3-B) to yield a cholesterol oligonucleotide conjugate (Fig, 3-C).

Fatty acids, alkylamines, alkyl alcohols and other lipids can be also added following procedures similar to those described for cholesterol.

Applications In addition to facilitate the transduction of oligonucleotides across cell membranes, another application of lipid-oligonucleotide conjugates is targeting of organs and specific cells. For instance, conjugation of an oligonucleotide and one cholesterol molecule to form a 3’-cholesterol-ODN results in a significant uptake by the liver. However, incorporation of another cholesterol to yield a 3’, 5’-bischolesteryl conjugate resulted in an almost quantitative uptake by the liver. An elegant example of the properties that steroids can confer to conjugates of DNA, PNA, and others oligonucleotides analogs can be found in the PNA-steroid dexamethasone conjugates that bind to the cytoplasmic glucocorticoid receptor (GR). The complex formed by the PNA-dexamethasone conjugate and the GR can then translocate from the cytoplasm into the nucleus.

Carbohydrate-oligonucleotide conjugates These conjugates can have the carbohydrate moiety linked directly to the oligonucleotide or can be linked to a lipid or a peptide/protein. Due to the diversity of carbohydrate receptors present on the cell’s surface, oligosaccharides are useful for targeting conjugates to specific cells. For example, mannose linked to ODNs allows targeting of the mannose receptors present on certain cells and the same approach has been used for galactose. ODNs conjugated to sucrose show a better stability against enzymatic degradation. Sugar-ODN conjugates can form hybrid duplexes to complementary DNA with a higher affinity than that observed with natural DNA. Conjugation of antisense ODNs to a neoglycoprotein in which the protein has several mannose-6-phosphate residues, is readily internalized by cells having a receptor for this ligand on their surfaces. In contrast to peptides and lipids, the number of carbohydrate-oligonucleotide conjugates is relatively small. Because sugars cannot translocate the cell membranes the sugar residues are usually present in combination with either lipids or peptides to target the lectins present of the cell’s surface.