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http://www.scq.ubc.ca/ Sequencing by Synthesis • Newer method to sequence whole genomes – Uses allyl protecting group: color PPPO DNA 3'-OH O + B Mixture of dNTPS O allyl polymerase O DNA color O P O O OH B O RXN STOPS! Pd0 (30 s, deprotects allyl moieties) O DNA O P O O OH OH B free to repeat Sequencing by Synthesis Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640 Copyright ©2006 by the National Academy of Sciences DNA/RNA Analogues • There have been several recent reports of the modification of oligonucleotides • Modifications have included: – Altering nitrogenous base structure – Employing different sugar structures – Modifying the backbone – Or in combination H2N N N O O N N O O OH(H) O P O NH O O N O O O H N OH(H) N O P O O O O N OH(H) N NH2 • Why make oligo analogues? – Structure/activity relationships (i.e., catalytic versatility) – Antisense technology – Insight into evolutionary process? • Synthetic oligos can be designed to bind with itself, DNA, RNA or all of the above • Effects: – H-bonding (base-pairing) – Other types of interactions (i.e. Van de Waals) – Overall shape (i.e., double helix? Hairpin loop?) • How do we examine interactions? – – – – Melting temp. Tm = 40.5 °C (DNA), 42.5 °C (RNA) NMR (as well other spectroscopic methods) Xray crystallography Calculations Antisense Technology • Employs a synthetic oligo that is complementary (antisense) to an mRNA sequence of interest • One of Two main effects can occur: – Translation arrest (no protein) – Recruits RNAase that degrades mRNA • Potential for therapeutic use – Vitravene (acts on CMV virus) Unnatural Base Pairs • Recall the natural base pairing (Watson-Crick) in DNA: D A H H N O H N N N sugar N sugar A N D N N O H A D H G C • If we change the number of or location of donor/acceptor groups, what interactions can occur? For example: Expand the “genetic alphabet” N • Tm measurements vary dramatically due to changes in H-bonding properties & hydrophobicity D D NH2 N N N N A N A N sugar N sugar NH2 Adenine D D H N H D H N H A H N N • DNA polymerase found to work with some modified bases (oligos) H N A N O N O N H D H D N N A N A sugar A A NH2 NH2 D N D sugar sugar • Such structures would be unlikely to form under prebiotic conditions NH2 Cytosine sugar Cytosine A D O N N NH2 N A N N N N sugar NH2 D guanine N sugar A O A Employing Novel Sugars Some examples: O • 5- vs 6- membered ring 5' Base O • Location of phosphodiester bond 1' 4' 2' 3' • Hydroxyl groups (# & stereochemistry) O • Position of base P OH(H) DNA/RNA O 6' 5' 4' O Base O OH 3' 1' 5' 4' O Base 1' 5' 4' 3' 2' O P -D-Xylopyranosyl NA O 6' O 3' 2' P Hexitol NA (HNA) Base O OH 1' 2' O P 6' OH Altropyranosyl-NA 5' O O Base 1' 4' O 3' 2' P Homo-NA O 6' 5' 4' O O Base 3' 2' P Hexitol NA (HNA) 1' • Adopts chair conformation in oligo • Forms helical duplex with RNA (Tm = + 3°C) & with DNA (Tm = + 3°C) • Adding more hydroxyl groups, increases affinity for RNA – Also increases thermal stability Modification of the Backbone • Peptide Nucleic Acid (PNA) – Aminoethyl glycine units linked by a peptide bond – achiral OH OH NH2 NH2 R O HN Base Base N R O O O HO O NH O R NH Base O HN P -O O O O Base R O O N NH R O HO O Base NH -O P O O O OH O Base HO N O CONH2 Protein PNA RNA • Duplex formation – PNA-DNA Tm = 68.8 °C (~20 °C higher than DNA-DNA!) – PNA-RNA Tm = 72.2 °C • PNA has the recognition properties of DNA (can carry genetic information) & has a higher stability than RNA and DNA • Stability? – Lack of phosphate groups → neutral backbone no electrostatic repulsion! – Not recognized by proteases • Did a PNA molecule precede RNA? – Has demonstrated numerous forms: hairpins, triplexes, etc – Simple in structure (i.e., achiral) & stable – Amino building blocks in primordial soup Another Modified Nucleic Acid - GNA OH glycol • Glycol nucleic acid OH • Can nucleic acid be made with such a simple sugar? – Is a ring structure important? – Minimum # of carbons? Synthesis of GNA O DMT-Cl O DMTO HO H N each base (protected) O CN R2N R2N P O P O HO CN Cl DMTO SN2 (inversion @ least hindered centre) N (Base) R & S GNA made DNA synthesizer DMTO N (Base) • Tested duplex formation: – Duplex formation Tm = 63 °C (22 ° higher than same DNA or RNA sequence!) – GNA more stable than DNA! – Demonstrates that cyclic sugar not necessary! • Was ribose in fact the sugar in the first pre-biotic nucleic acids? • Is GNA a pre-RNA candidate? – A glycerol derivative, which is related to triose – Recall, we looked at 3C sugars (i.e., glyceraldehyde) in relation to the prebiotic formation of sugars An example of chemical biology: • Uses biological concepts, DNA structure • Uses chemical ideas → conformation & functional groups • Uses chemical synthesis principles • Makes a non-natural molecule with novel properties • Relates those properties to the natural system • Problem: Still difficult to predict and analyze singlestranded oligonucleotide structures