Section C - Properties of Nucleic Acids

Contents C1 Nucleic Acid Structure Bases, Nucleosides, Nucleotides, Phosphodiester bonds, DNA/RNA sequence, DNA double helix, A, B and Z helices, RNA secondary structure, Modified nucleic acids C2 Chemical and Physical Properties of Nucleic Acids Stability of Nucleic Acids, Effect of acid, Effect of alkali, Chemical denaturation, Viscosity, Buoyant density C3 Spectroscopic Properties of Nucleic Acids UV absorption, Hypochromicity, Quantization of nucleic acids, Purity of DNA, Thermal denaturation, Renaturation C4 DNA supercoiling Closed-circular, Supercoiling, Topoisomer, Twist and writhe, Intercalators, Energy of supercoiling, Topoisomerases

C1 Nucleic Acid Structure — Bases Bicyclic Purine Monocyclic Pyrimidine

C1 Nucleic Acid Structure — Nucleosides Glycosidic (glycoside, glycosylic) bond (糖苷键)

C1 Nucleic Acid Structure — Nucleotides A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached.

BASES NUCLEOSIDES NUCLEOTIDES Adenine (A) Adenosine Adenosine 5’-triphosphate (ATP) Deoxyadenosine Deoxyadenosine 5’-triphosphate (dATP) Guanine (G) Guanosine Guanosine 5’-triphosphate (GTP) Deoxyguanosine Deoxy-guanosine 5’-triphosphate (dGTP) Cytosine (C) Cytidine Cytidine 5’-triphosphate (CTP) Deoxycytidine Deoxy-cytidine 5’-triphosphate (dCTP) Uracil (U) Uridine Uridine 5’-triphosphate (UTP) Thymine (T) Thymidine/ Deoxythymidie Thymidine/deoxythymidie 5’-triphosphate (dTTP)

C1 Nucleic Acid Structure — Phosphodiester bonds

C1 Nucleic Acid Structure — DNA/RNA sequence

C1 Nucleic Acid Structure — DNA double helix Watson and Crick , 1953 The genetic material of all organisms except for some viruses The foundation of the molecular biology Watson Crick

Essential for replicating DNA and transcribing RNA Two separate strands Antiparellel (5’3’ direction) Complementary (sequence) Base pairing: hydrogen bonding that holds two strands together 3’ 5’ Sugar-phosphate backbones (negatively charged): outside Planner bases (stack one above the other): inside 3’ 5’

Helical turn: 10 base pairs/turn 34 Ao/turn

Base pairing

C1 Nucleic Acid Structure — A, B and Z helices

C1 Nucleic Acid Structure — RNA secondary structure Single stranded nucleic acid Secondary structure are formed some time Globular tertiary structure are important for many functional RNAs, such as tRNA, rRNA and ribozyme RNA Forces for secondary and tertiary structure: intramolecular hydrogen bonding and base stacking.

Conformational (构象) variability of RNA is important for the much more diverse roles of RNA in the cell, when compared to DNA. Structure and Function correspondence of protein and nucleic acids Protein Nucleic Acids Fibrous protein Globular protein Helical DNA Globular RNA Structural proteins Enzymes, antibodies, receptors etc Genetic information maintenance Ribozymes Transfer RNA (tRNA) Signal recognition etc.

COMMON SECONDARY STRUCTURE MOTIFS

C1 Nucleic Acid Structure — Modified nucleic acids 1. Methylation: (N-6position of adenine ,4-amino group and 5-position of cytosine) Restriction modification 2. Base mismatch: Modifications correspond to numbers of specific roles. We will discuss them in some related topics. For example, methylation of A and C to can avoid restriction digestion of endogenous DNA sequence.

Deoxyribonucleic acids Nucleic acid analogues Types of nucleic acids Constituents Nucleobases • Nucleosides • Nucleotides • Deoxynucleotides Ribonucleic acids RNA • mRNA (pre-mRNA/hnRNA) • tRNA • rRNA • aRNA • gRNA • miRNA • ncRNA • piRNA • shRNA • siRNA • snRNA • snoRNA • stRNA • ta-siRNA • tmRNA Deoxyribonucleic acids DNA • cDNA • cpDNA • gDNA • msDNA • mtDNA Nucleic acid analogues GNA • LNA • PNA • TNA • morpholino Cloning vectors phagemid • plasmid • lambda phage • cosmid • P1 phage • fosmid • BAC • YAC • HAC

2. Stacking interaction/hydrophobic interaction C2 Chemical and Physical Properties of Nucleic Acids — Stability of Nucleic Acids Hydrogen bonding Does not normally contribute the stability of nucleic acids or protein Contributes to specific structures of these macromolecules. For example, a-helix, b-sheet, DNA double helix, RNA secondary structure 2. Stacking interaction/hydrophobic interaction Between aromatic base pairs/bases contribute to the stability of nucleic acids. It is energetically favorable for the hydrophobic bases to exclude waters and stack on top of each other This stacking is maximized in double-stranded DNA

C2 Chemical and Physical Properties of Nucleic Acids — Effect of acid Strong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphate pH 3-4 : apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ], can be generated by formic acid

RNA hydrolyzes at higher pH because of 2’-OH groups in RNA C2 Chemical and Physical Properties of Nucleic Acids — Effect of alkali RNA hydrolyzes at higher pH because of 2’-OH groups in RNA

High pH (> 7-8) has subtle (small) effects on DNA structure High pH changes the tautomeric (互变异构)state of the bases keto form enolate form Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation

Strong acid + high temperature: C2 Chemical and Physical Properties of Nucleic Acids — Chemical denaturation Strong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphate Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure

C2 Chemical and Physical Properties of Nucleic Acids — Viscosity Buoyant density Physical properties Reasons for the DNA high viscosity High axial ratio Relatively stiff Applications: Long DNA molecules can easily be shortened by shearing force. Remember to avoid shearing problem when isolating very large DNA molecule.

C2 Chemical and Physical Properties of Nucleic Acids — Buoyant density 1.7 g cm-3, a similar density to 8M CsCl Purifications of DNA: equilibrium density gradient centrifugation Protein floats RNA pellets at the bottom

C3 Spectroscopic Properties of Nucleic Acids — UV absorption Nucleic acids absorb UV light due to the aromatic bases The wavelength of maximum absorption by both DNA and RNA is 260 nm Applications: detection, quantitation, assessment of purity (A260/A280) Quantitation of nucleic acids: Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20；ssDNA and RNA, 25

C3 Spectroscopic Properties of Nucleic Acids — Hypochromicity Hypochromicity: caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide

C3 Spectroscopic Properties of Nucleic Acids — Quantitation of nucleic acids Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20 ssDNA and RNA, 25 The values for ssDNA and RNA are approximate The values are the sum of absorbance contributed by the different bases (e.g. purines > pyrimidines) The absorbance values also depend on the amount of secondary structures due to hypochromicity

C3 Spectroscopic Properties of Nucleic Acids — Purity of DNA A260/A280 dsDNA--1.8 pure RNA--2.0 protein--0.5

C3 Spectroscopic Properties of Nucleic Acids — Thermal denaturation Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.

C3 Spectroscopic Properties of Nucleic Acids — Renaturation Rapid cooling: only allow the formation of local base paring. Absorbance is slightly decreased. Slow cooling: whole complementation of dsDNA. Absorbance decreases greatly and cooperatively. Annealing: base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction) Hybridization: renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)

C4 DNA supercoiling — Closed-circular cccDNA: Covalently closed circular Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled.

C4 DNA supercoiling — Supercoiling Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding of double helix. Lk: linking number Lk。: the linking number for a relaxed closed

C4 DNA supercoiling — Topoisomer A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.

C4 DNA supercoiling — Twist and writhe Supercoiling is partitioned geometrically into a change in twist, the local winding up or unwinding of the double helix, and a change in writhe, the coiling of the helix axis up itself. Twist and writhe are interconvertible according to the equation: ΔLK=ΔTω+ΔWr

C4 DNA supercoiling — Intercalators Ethidium bromide (intercalator): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.

C4 DNA supercoiling — Energy of supercoiling Negatively supercoiled DNA has a high torsional energy, which facilitates the untwisting of DNA helix and can drive processes which require the DNA to be unwound. Such as transcription initiation or replication.

C4 DNA supercoiling — Topoisomerases Topoisomerases exist in cell to regulate the level of supercoiling of DNA molecules. Type I topoisomerase: breaks one strand and change the linking number in steps of ±1. TypeII topoisomerase: breaks both strands and change the linking number in steps of ±2. Gyrase: introduce the negative supercoiling (resolving the positive one and using the energy from ATP hydrolysis.

Multiple choice questions 1．The sequence 5'-AGTCTGACT-3' in DNA is equivalent to which sequence in RNA? A 5'-AGUCUGUGACU -3' B 5' -UGTCTGUTC -3' C 5' -UCAGUCUGA-3' D 5'- AGUCAGACU-3'

2. Which of the following correctly describes A-DNA? A a right-handed antiparallel double helix with 10 bp/turn and bases lying perpendicular to the helix axis. B a left-handed antiparallel double-helix with 12 bp/turn formed from alternating pyrimidine-purine sequences. C a right-handed antiparallel double helix with 11 bp/turn and bases tilted with respect to the helix axis. D a globular structure formed by short intramolecular helices formed in a single-strand nucleic acid. 3. Denaturation of double stranded DNA involves . A preakage into short double-stranded fragments. B separation into single strands. C hydrolysis of the DNA backbone. D cleavage of the bases from the sugar-phosphate backbone.

4. Which has the highest absorption per unit mass at a wavelength of 260 nm? A double-stranded DNA. B mononucleotides. C RNA. D protein. 5. Type I DNA topoisomeraes . A change linking number by士2 B require ATP. C break one strand of a DNA double helix. D are the target of antibacterial drugs.

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