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In biochemistry, intercalation is the insertion of molecules between the planar bases of deoxyribonucleic acid (DNA). This process is used as a method for analyzing DNA and it is also the basis of certain kinds of poisoning.
There are several ways molecules (in this case, also known as ligands ) can interact with DNA. Ligands may interact with DNA by covalently binding, electrostatically binding, or intercalating. [1] Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA. These ligands are mostly polycyclic, aromatic, and planar, and therefore often make good nucleic acid stains. Intensively studied DNA intercalators include berberine, ethidium bromide, proflavine, daunomycin, doxorubicin, and thalidomide. DNA intercalators are used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells. Examples include doxorubicin (adriamycin) and daunorubicin (both of which are used in treatment of Hodgkin's lymphoma), and dactinomycin (used in Wilm's tumour, Ewing's Sarcoma, rhabdomyosarcoma).
Metallointercalators are complexes of a metal cation with polycyclic aromatic ligands. The most commonly used metal ion is ruthenium(II), because its complexes are very slow to decompose in the biological environment. Other metallic cations that have been used include rhodium(III) and iridium(III). Typical ligands attached to the metal ion are dipyridine and terpyridine whose planar structure is ideal for intercalation. [2]
In order for an intercalator to fit between base pairs, the DNA must dynamically open a space between its base pairs by unwinding. The degree of unwinding varies depending on the intercalator; for example, ethidium cation (the ionic form of ethidium bromide found in aqueous solution) unwinds DNA by about 26°, whereas proflavine unwinds it by about 17°. This unwinding causes the base pairs to separate, or "rise", creating an opening of about 0.34 nm (3.4 Å). This unwinding induces local structural changes to the DNA strand, such as lengthening of the DNA strand or twisting of the base pairs. These structural modifications can lead to functional changes, often to the inhibition of transcription and replication and DNA repair processes, which makes intercalators potent mutagens. For this reason, DNA intercalators are often carcinogenic, such as the exo (but not the endo) 8,9 epoxide of aflatoxin B1 and acridines such as proflavine or quinacrine.
Intercalation as a mechanism of interaction between cationic, planar, polycyclic aromatic systems of the correct size (on the order of a base pair) was first proposed by Leonard Lerman in 1961. [3] [4] [5] One proposed mechanism of intercalation is as follows: In aqueous isotonic solution, the cationic intercalator is attracted electrostatically to the surface of the polyanionic DNA. The ligand displaces a sodium and/or magnesium cation present in the "condensation cloud" of such cations that surrounds DNA (to partially balance the sum of the negative charges carried by each phosphate oxygen), thus forming a weak electrostatic association with the outer surface of DNA. From this position, the ligand diffuses along the surface of the DNA and may slide into the hydrophobic environment found between two base pairs that may transiently "open" to form an intercalation site, allowing the ethidium to move away from the hydrophilic (aqueous) environment surrounding the DNA and into the intercalation site. The base pairs transiently form such openings due to energy absorbed during collisions with solvent molecules.
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific hydrogen bonding patterns, "Watson–Crick" base pairs allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes.
Mutagenesis is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. A mutagen is a mutation-causing agent, be it chemical or physical, which results in an increased rate of mutations in an organism's genetic code. In nature mutagenesis can lead to cancer and various heritable diseases, and it is also a driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.
Acridine is an organic compound and a nitrogen heterocycle with the formula C13H9N. Acridines are substituted derivatives of the parent ring. It is a planar molecule that is structurally related to anthracene with one of the central CH groups replaced by nitrogen. Like the related molecules pyridine and quinoline, acridine is mildly basic. It is an almost colorless solid, which crystallizes in needles. There are few commercial applications of acridines; at one time acridine dyes were popular, but they are now relegated to niche applications, such as with acridine orange. The name is a reference to the acrid odour and acrid skin-irritating effect of the compound.
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, tests have shown it to have low mutagenicity without metabolic activation.
Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used together with other chemotherapy agents. Doxorubicin is given by injection into a vein.
Daunorubicin, also known as daunomycin, is a chemotherapy medication used to treat cancer. Specifically it is used for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and Kaposi's sarcoma. It is administered by injection into a vein. A liposomal formulation known as liposomal daunorubicin also exists.
In molecular biology, the term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its tertiary structure. The structure was discovered by Maurice Wilkins, Rosalind Franklin, her student Raymond Gosling, James Watson, and Francis Crick, while the term "double helix" entered popular culture with the 1968 publication of Watson's The Double Helix: A Personal Account of the Discovery of the Structure of DNA.
In chemistry, a non-covalent interaction differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule. The chemical energy released in the formation of non-covalent interactions is typically on the order of 1–5 kcal/mol. Non-covalent interactions can be classified into different categories, such as electrostatic, π-effects, van der Waals forces, and hydrophobic effects.
Cetrimonium bromide, also known with the abbreviation CTAB, is a quaternary ammonium surfactant with a condensed structural formula [(C16H33)N(CH3)3]Br.
The tropylium ion or cycloheptatrienyl cation is an aromatic species with a formula of [C7H7]+. Its name derives from the molecule tropine from which cycloheptatriene (tropylidene) was first synthesized in 1881. Salts of the tropylium cation can be stable, even with nucleophiles of moderate strength e.g., tropylium tetrafluoroborate and tropylium bromide (see below). Its bromide and chloride salts can be made from cycloheptatriene and bromine or phosphorus pentachloride, respectively.
Cation–π interaction is a noncovalent molecular interaction between the face of an electron-rich π system (e.g. benzene, ethylene, acetylene) and an adjacent cation (e.g. Li+, Na+). This interaction is an example of noncovalent bonding between a monopole (cation) and a quadrupole (π system). Bonding energies are significant, with solution-phase values falling within the same order of magnitude as hydrogen bonds and salt bridges. Similar to these other non-covalent bonds, cation–π interactions play an important role in nature, particularly in protein structure, molecular recognition and enzyme catalysis. The effect has also been observed and put to use in synthetic systems.
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.
Leonard Solomon Lerman was an American scientist most noted for his work on DNA.
An electrophoretic color marker is a chemical used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. The color markers are made up of a mixture of dyes that migrate through the gel matrix alongside the sample of interest. They are typically designed to have different mobilities from the sample components and to generate colored bands that can be used to assess the migration and separation of sample components.
In chemistry, π-effects or π-interactions are a type of non-covalent interaction that involves π systems. Just like in an electrostatic interaction where a region of negative charge interacts with a positive charge, the electron-rich π system can interact with a metal, an anion, another molecule and even another π system. Non-covalent interactions involving π systems are pivotal to biological events such as protein-ligand recognition.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. The intrinsic DNA fluorescence is very weak.Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Nucleic acid tertiary structure is the three-dimensional shape of a nucleic acid polymer. RNA and DNA molecules are capable of diverse functions ranging from molecular recognition to catalysis. Such functions require a precise three-dimensional structure. While such structures are diverse and seemingly complex, they are composed of recurring, easily recognizable tertiary structural motifs that serve as molecular building blocks. Some of the most common motifs for RNA and DNA tertiary structure are described below, but this information is based on a limited number of solved structures. Many more tertiary structural motifs will be revealed as new RNA and DNA molecules are structurally characterized.
DNA-binding metallo-intercalators are positively charged, planar, polycyclic, aromatic compounds that unwind the DNA double helix and insert themselves between DNA base pairs. Metallo-intercalators insert themselves between two intact base pairs without expelling or replacing the original nitrogenous bases; the hydrogen bonds between the nitrogenous bases at the site of intercalation remain unbroken. In addition to π-stacking between the aromatic regions of the intercalator and the nitrogenous bases of DNA, intercalation is stabilized by van der Waals, hydrophobic, electrostatic, and entropic interactions. This ability to bind to specific DNA base pairs allows for potential therapeutic applications of metallo-intercalators.
i-motif DNA, short for intercalated-motif DNA, are cytosine-rich four-stranded quadruplex DNA structures, similar to the G-quadruplex structures that are formed in guanine-rich regions of DNA.
SYBR Gold is an asymmetrical cyanine dye. It can be used as a stain for double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold is the most sensitive fluorescent stain of the SYBR family of dyes for the detection of nucleic acids. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific
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