Table of Contents Why DNA Computing? The Structure of DNA DNA - PDF document

Table of Contents Æ Why DNA Computing? Æ The Structure of DNA DNA Computing Æ Operations on DNA Molecules Æ Reading DNA Æ Example of a Molecular Computer Information Processing with DNA Molecules Christian Jacob Why DNA Computing? Challenges of DNA Computing Æ From silico to carbon. Æ Biochemical techniques are not yet From microchips to DNA molecules. sufficiently sophisticated or accurate. Æ Limits to miniaturization with current Æ Compare Charles Babbage´s „Analytical Engine“ (1810-1820) computer technologies. Æ Information processing capabilities of organic molecules ... Æ replace digital switching primitives, Æ enable new computing paradigms. Still: Why DNA Computing? Key Features of DNA Computing Æ Massive parallelism of DNA strands Æ Further reasons to investigate DNA computing: Æ high density of information storage Æ support for standard computation Æ ease of constructing many copies Æ better understanding of how nature Æ Watson-Crick complementarity computes Æ new data structures (molecules) Æ feature provided „for free“ Æ new operations Æ universal twin shuffle language l cut, paste, adjoin, insert, delete, ... Æ new computability models.

Table of Contents The Structure of DNA Æ Why DNA Computing? Æ DNA is a polymer („large“ molecule). Æ The Structure of DNA Æ DNA is strung together from monomers („small“ mols.): deoxyribonucleotides. Æ Operations on DNA Molecules Æ Reading DNA Æ DNA = Deoxyribo Nucleic Acid Æ Example of a Molecular Computer Æ DNA supports two key functions for life: Æ coding for the production of proteins, Æ self-replication. Structure of a DNA Monomer Chemical Structure of a Nucleotide Æ Each deoxyribonucleotide consists of three components: Æ a sugar — deoxyribose Æ five carbon atoms: 1´ to 5´ Æ hydroxyl group (OH) attached to 3´ carbon Æ a phosphate group Æ a nitrogenous base. Linking of Nucleotides Structure of a DNA Monomer (2) Æ DNA nucleotides differ only by their Æ The DNA monomers can link in two ways: bases (B): Æ Phosphodiester bond Æ Hydrogen bond Æ purines Æ Adenine A Æ Guanine G Æ pyrimidines Æ Cytosine C Æ Thymine T

Linking of Nucleotides Linking of Nucleotides Phosphodiester Bond Phosphodiester Bond Æ The 5´-phosphate group of one nucleotide is joined with the 3´-hydroxyl group of the other Æ strong (covalent) bond Æ directionality: 5´—3´ or 3´—5´ Linking of Nucleotides Linking of Nucleotides Hydrogen Bond Hydrogen Bond Æ The base of one nucleotide interacts with the base of another Æ base pairing (weak bond) l A — T (2 hydrogen bonds) l C — G (3 hydrogen bonds) Æ Watson-Crick complementarity l James D. Watson l Francis H. C. Crick l deduced double-helix structure of DNA in 1953 l Nobel Prize (1962) DNA Double Helix Table of Contents Æ Longer streches keep the double Æ Why DNA Computing? strands together through the Æ The Structure of DNA cumulative effect (the sum) of hydrogen bonds. Æ Operations on DNA Molecules Æ Reading DNA Æ Dense packing: l Bacteria: DNA molecule is 10,000 times Æ Example of a Molecular Computer longer than the host cell l Eucaryotes: „hierarchical“ packing

Operations on DNA Molecules Separating and Fusing DNA Strands Æ Separating and fusing DNA strands Æ Denaturation: separating the single strands without breaking them Æ Lengthening of DNA Æ weaker hydrogen than phosphodiester Æ Shortening DNA bonding Æ Cutting DNA Æ heat DNA (85° - 90° C) Æ Multiplying DNA Æ Renaturation: Æ slowly cooling down Æ annealing of matching, separated strands Enzymes Lengthening DNA Machinery for Nucleotide Manipulation Æ Enzymes are proteins that catalyze Æ DNA polymerase enzymes chemical reactions. add nucleotides to a DNA molecule Æ Enzymes speed up chemical reactions Æ Requirements: extremely efficiently (speedup: 1012) Æ single-stranded template Æ Enzymes are very specific. Æ primer, Æ Nature has created a multitude of l bonded to the template enzymes that are useful in processing l 3´-hydroxyl end available for DNA. extension l Note: Terminal transferase needs no primer. Shortening DNA Shortening DNA Æ DNA nucleases are enzymes Æ DNA nucleases are enzymes that degrade DNA. that degrade DNA. Æ DNA exonucleases Æ DNA exonucleases l cleave (remove) nucleotides one at l cleave (remove) nucleotides one at a time from the ends of the strands a time from the ends of the strands l Example: Exonuclease III l Example: Bal31 3´-nuclease removes nucleotides from both degrading in 3´-5´direction strands

Cutting DNA Multiplying DNA Æ DNA nucleases are enzymes Æ Amplification of a „small“ amount of a specific that degrade DNA. DNA fragment, lost in a huge amount of other pieces. Æ DNA endonucleases Æ „Needle in a haystack“ l destroy internal phosphodiester bonds Æ Solution: PCR = Polymerase Chain Reaction l Example: S1 cuts only single strands or within Æ devised by Karl Mullis in 1985 single strand sections Æ Nobel Prize Æ Restriction endonucleases Æ a very efficient molecular Xerox machine l much more specific l cut only double strands l at a specific set of sites (EcoRI) PCR PCR Step 0: Initialization Step 1: Denaturation Æ Start with a solution containing Æ Solution heated close to boiling the following ingredients: temperature. l the target DNA molecule Æ Hydrogen bonds between the l primers double strands are separated (synthetic oligonucleotides), into single strand molecules. complementary to the terminal sections l polymerase, heat resistant l nucleotides PCR PCR Step 2: Priming Step 3: Extension Æ The solution is cooled down (to Æ The solution is heated again (to about 55° C). about 72° C). Æ Primers anneal to their Æ A polymerase will extend the complementary borders. primers, using nucleotides available in the solution. Æ Two complete strands of the target DNA molecule are produced.

PCR Table of Contents Efficient Xeroxing: 2n copies after n steps Æ Why DNA Computing? Æ The Structure of DNA Step 1 Æ Operations on DNA Molecules Step 2 Æ Reading DNA Æ Example of a Molecular Computer Step 3 Step 4 Step 5 Measuring the Length of DNA Molecules Schematic representation of gel electrophoresis Gel Electrophoresis Radioactive marker Æ DNA molecules are negatively charged. Æ Placed in an electric field, they will move towards the positive electrode. Æ The negative charge is proportional to the length of the DNA molecule. Ethidium bromide marker Æ The force needed to move the molecule is proportional to its length. Æ A gel makes the molecules move at different speeds. Æ DNA molecules are invisible, and must be marked (ethidium bromide, radioactive) Sequencing a DNA Molecule Sequencing — Part 1 Æ Sequencing: Æ Objective Æ reading the exact sequence of nucleotides Æ We want to sequence a single stranded molecule comprising a given DNA molecule � . Æ based on Æ Preparation l the polymerase action of extending a primed single Æ We extend � at the 3´ end by a short (20 bp) stranded template sequence � , which will act as the W-C complement l nucleotide analogues for the primer compl ( � ). l chemically modified l Usually, the primer is labelled (radioactively, or marked l e.g., replace 3´-hydroxyl group (3´-OH) by 3´- fluorescently) hydrogen atom (3´-H) Æ This results in a molecule � ´= 3´- �� . l dideoxynucleotides: - ddA, ddT, ddC, ddG l Sanger method, dideoxy enzymatic method

Sequencing — Part 2 Reaction in Tube A Æ 4 tubes are prepared: Æ The polymerase enzyme l Tube A, Tube T, Tube C, Tube G extends the primer of � ´, using l Each of them contains the nucleotides present in l � molecules Tube A: l primers, compl ( � ) ddA, A, T, C, G. l polymerase Æ using only A, T, C, G: l nucleotides A, T, C, and G. l � ´ is extended to the full duplex. l Tube A contains a limited amount of ddA. l Tube T contains a limited amount of ddT. Æ using ddA rather than A: l Tube C contains a limited amount of ddC. l complementing will end at the l Tube G contains a limited amount of ddG. position of the ddA nucleotide. Resulting Sequences in Tubes Final Reading of the Strands Æ Tube A: Æ Tube C: Æ We read: Æ TCATGCACTGCG Æ TCATGCACTGCG Gel Æ T Æ TC Æ TCA electrophoresis: Æ TC Æ TCATGC Æ TCA Æ TCATGCA Æ TCATGCAC Æ TCAT Æ TCATG Æ TCATGCACTGC Æ Tube T: Æ Tube A: Æ TCATGC Æ Tube C: Æ TCATGCACTGCG Æ TCATGCA TCATGCACTGCG l TCATGCACTGCG Æ Tube G: l Æ TCATGCAC TCA l TC Æ T l Æ TCATGCACTGCG l TCATGC TCATGCA Æ TCATGCACT l Æ TCAT l TCATGCAC Æ TCATG Æ TCATGCACTG Æ Tube T: l TCATGCACTGC Æ TCATGCACT Æ TCATGCACTGC Æ TCATGCACTG TCATGCACTGCG l Æ TCATGCACTGCG Æ Tube G: T l l TCATGCACTGCG TCAT l l TCATG TCATGCACT l l TCATGCACTG Table of Contents Adleman´s Experiment Æ Why DNA Computing? Æ In 1994 Leonard M. Adleman showed how to solve the Hamilton Path Problem, using DNA Æ The Structure of DNA computation. Æ Operations on DNA Molecules Æ Reading DNA Æ Hamiltonian Path Problem: Æ Let G be a directed graph with designated input Æ Example of a Molecular Computer and output vertices, vin and vout. Æ Find a (Hamiltonian) path from vin to vout that involves every vertex in G exactly once.