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Facoltà di Scienze Agrarie e Alimentari Università degli Studi di Milano
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Chemistry of Nucleic Acids
Code: G6108-
Teacher:  Enzio Maria Ragg
CFU subdivision: Lectures: 5
Practices in laboratory:  1
Basic aims:  This Course is for students interested in getting a wider understanding of the molecular aspects of Biotechnology. Different aspects of the Chemistry of Nucleic Acids will be covered, in particular underlying its implications in the biotechnological, food and pharmaceutical research fields.
Teaching materials will be given exclusively in English. In case, lessons can also be given in english
Acquired skills:  Basic knowledge of structure and reactivity of nucleic acids and of the fundamental chemical methodologies of extraction, purification and characterization of natural organic substances. Familiarity of experimental and computational methodologies for the study of ligand-DNA interactions
Course contents:  Structure and reactivity of nucleic acids and their constituents. Methods of oligonucleotide chemical synthesis. Topological forms of DNA. Biological meaning of non -conventional structures of DNA and RNA. Spectroscopic and computational methods of structural analysis. Mechanism of DNA-xenobiotic interactions. Natural and synthetic carcinogens.
Laboratory tutorials: Alkaloid extraction. Measurement of DNA constant affinity with DNA. Measurement of ionization constants by NMR. Prediction of folding and thermal stability of RNA-oligomers. Analysis of crystallographic and NMR structures of oligonucleotides, free and in complex with peptides or covalently linked organic molecules
Program:  Class lectures ( 5 CFU):
Structure and reactivity of nucleic acids: Structure and reactivity of aromatic bases and ribose units. Acid-base equilibria. Hydrogen bond donor and acceptor sites. Nucleophylic sites. Reaction mechanism with electrophylic, alkylating and radical species. The Maxham and Gilbert sequencing method. Conformational analysis of ribose: E (envelope) and T (twist) forms. Structural parameters of the double helix (A- and B-DNA): helical twist, propeller twist; distance of neighbour bases, helical pitch, geometry of minor and major grooves. Thermodinamics of double helix and hairpins. Temperature of melting measurements by UV-vis spectroscopy. Nearest-neighbour predictive models. Role of dangling ends in helix stability.
Methods of conformational analysis. NMR spettroscopy. The physical basis of NMR. Spin-active nuclei: 1-H, 13-C, 31-P. The chemical-shift. Resonance fine structure: spin-spin coupling. Molecular modelling: analysis of crystallographic structures; molecular mechanics: force-field definitions.
DNA-xenobiotic interactions. Mechanisms of interaction: intercalation, minor groove interactions, alkylation, electrostatic interactions. Analytical methods: Tm variations by UV measurements; footprinting experiments of labeled oligonucleotides with radioactive probes. Dissociation constants measurements (with numerical calculations).
Carcinogenic agents. Carcinogenic molecules of natural and synthetic origin. Nitrogen and sulphur mustards. Polycyclic Aromatic Hydrocarbons. Amines and nitrosoamines. Aflatoxins. Mechanisms of action.
Non conventional structures of NA Hydrogen bonding in non-conventional base pairs Tripkle helices and G-quadruplexes. Analysis of stabilizing factors: propensity of single residues, pH, specific ionic interactions.
Topological forms of DNA. DNA bending in homopurinic tracts with regular sequence (e.g. A5N5). Electrophoretic mobility. Supercoiled forms of DNA: definition of Ln (linking), Wr ( Writhe) and Tw (Twist) parameters. Supercoil measurements through gel-elettrophoresis mobility. Modes of function of topoisomerases.

Laboratory tutorials (1 CFU):

• DNA-small molecule interactions by equilibrium dyalisis
• Structural analysis on a computer by access to crystallographic Data Banks; structure generation and interaction studies with small molecules and proteins with the aid of specific molecular mechanics and dynamics simulation programs
• Free energy, enthalpy and entropy variations of double helical oligonucleotides, with or without intercalators, comparison with spectroscopic data (UV or NMR)
• Ionization costants measurements by 31P, 1H e 13C-ch. shift measurements.
Prerequisites:  Basic knowledge of informatics. Familiarity with the most common Organic Chemistry Laboratory techniques
Preparatory instructions:  Organic Chemistry with Laboratory Practice. Biochemistry.
Learning materials:  Teaching material will be directly given by the teacher.
Articles from scientific journals (ARIEL site)
Other info:  Oral examination in dates to be agreed with the teacher.
Discussion on topics related to the class lectures and laboratory tutorials. A PDB code of a ligand-DNA complex will be agreed by teacher and student. At the day of examination the student will describe the system on a detailed level with the aid of representative pictures. He will be asked to describe in particular:
a. oligonucleotide sequence
b. molecular structure of the ligand
c. points of contact between ligand and DNA
Program of Chemistry of Nucleic Acids (pdf version)
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