Properties of Nucleic Acids Molecular Biology SECTION C Properties of Nucleic Acids C1 Nucleic Acid Structure C2 Chemical and Physical Properties of Nucleic Acids C3 Spectroscopic and Thermal Properties of Nucleic Acids C4 DNA Supercoiling

C1 Nucleic Acid Structure-1 Molecular Biology C1 Nucleic Acid Structure-1 Bases Bicyclic Purines: Monocyclic pyrimidine: Thymine (T) is a 5-methyluracil (U)

C1 Nucleic Acid Structure-2 Molecular Biology C1 Nucleic Acid Structure-2 Nucleosides The structures of pentose sugar

Adenosine, guanosine, cytidine, thymidine, uridine Molecular Biology The bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside Adenosine, guanosine, cytidine, thymidine, uridine

Nucleotides C1 Nucleic Acid Structure-3 Molecular Biology C1 Nucleic Acid Structure-3 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. Phosphate ester bonds Deoxyribonucleotides (containing deoxyribose) Ribonucleotides (containing ribose)

Molecular Biology

Brief Summary Adenine (A) Guanine (G) Cytosine (C) Uracil (U) Molecular Biology Brief Summary 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-4 Phosphodiester bonds and Sequence Molecular Biology C1 Nucleic Acid Structure-4 Phosphodiester bonds and Sequence Primary sequence: 5’end: may or may not have any attached phosphate groups. 3’ end: is most likely to be a free hydroxyl group.

C1 Nucleic Acid Structure-5 Molecular Biology C1 Nucleic Acid Structure-5 DNA double helix Deduced by James Watson and Francis Crick in 1953. DNA is the genetic material of all organisms except for some viruses. Essential for replicating DNA and transcribing RNA The foundation of the molecular biology

Two separate strands Antiparellel (5’3’ direction) Molecular Biology Two separate strands Antiparellel (5’3’ direction) Complementary (sequence) Base pairing: hydrogen bonding that holds two strands together. The negatively charged Sugar-phosphate backbones are on the outside of helix. Planar bases stack one above the other in the center (inside) of helix.

Molecular Biology Base pairing G:C A:T

Molecular Biology Helical turn: 10 base pairs/turn 3.4 nm/turn

Molecular Biology

C1 Nucleic Acid Structure-6 Molecular Biology C1 Nucleic Acid Structure-6 A, B and Z helices A-form B-form Z-form

C1 Nucleic Acid Structure-7 Molecular Biology C1 Nucleic Acid Structure-7 RNA structure Normally occurs as single stranded molecule 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.

Molecular Biology Secondary structure tRNA Tertiary structure

Molecular Biology Conformational variability of RNA is reflected in the more diverse roles of RNA in the cell, when compared to DNA. Comparison of Structure and Function of protein and nucleic acids Protein Nucleic Acids Fibrous protein Globular protein Double Helical DNA Globular RNA Structural proteins Enzymes, antibodies, receptors etc Genetic material Ribozymes Transfer RNA (tRNA) Signal recognition etc.

C1 Nucleic Acid Structure-8 Modified Nucleic Acids Molecular Biology C1 Nucleic Acid Structure-8 Modified Nucleic Acids The chemical modification of bases or nucleotides in nucleic acids is widespread, and has a number of specific roles. In cellular DNA, the modifications are restricted to the methylation of the N-6 position of adenine and the 4-amino group and the 5-position of cytosine. These methylations have a role in restriction modification (see Topic G3), base mismatch repair (see Topic F3) and eukaryotic genome structure (see Topic D3). A much more diverse range of modifications occurs in RNA after transcription, which again reflects the different roles of RNA in the cell. These are considered in more detail in Topics O3 and P2。

C2 Chemical and Physical Properties of Nucleic Acids Molecular Biology C2 Chemical and Physical Properties of Nucleic Acids

Stability of Nucleic Acids Molecular Biology Stability of Nucleic Acids Hydrogen bonding Contributes to specific structures of nucleic acids or protein. For example, a-helix, b-sheet, DNA double helix, RNA secondary structures H-bonds within a structure does not normally confer the stability, that is to say, it does not contribute the overall stability of these secondary structures 2. Stacking interaction/hydrophobic interaction between aromatic base pairs contribute to the stability of nucleic acids. Even in single-stranded DNA, the bases have a tendency to stack on top of each other, but this stacking is maximized in double-stranded DNA It is energetically favorable to exclude water altogether from pairs of such surfaces by stacking them together

Molecular Biology Effect of Acid In strong acid and at elevated temperatures: are hydrolyzed (水解) completely to bases, ribose or deoxyribose, and phosphate (e.g., perchloric acid (HClO4) at > 100°C) In more dilute mineral acid, for example at pH 3–4, the most easily hydrolyzed bonds are selectively broken. E.g., glycosylic bonds attaching purine bases to the ribose ring are broken by formic acid. Application: the basis of the chemical DNA sequencing method developed by Maxam and Gilbert.

Molecular Biology Effect of Alkali-DNA Increasing pH (> 7-8) has more subtle effects on DNA structure The effect of alkali is to change the tautomeric (互变异构)state of the bases keto form enolate form keto form enolate form 3. This affects the specific hydrogen bonding between the base pairs, with the result that the double-stranded structure of the DNA breaks down; that is the DNA becomes denatured .

Effect of Alkali-RNA RNA is unstable at higher pH Molecular Biology Effect of Alkali-RNA RNA are hydrolyzed at higher pH because of the present of 2’-OH group in RNA HO Alkali 2’, 3’-cyclic phosphodiester RNA is unstable at higher pH

Chemical Denaturation Molecular Biology Chemical Denaturation A number of chemical agents can cause the denaturation of DNA or RNA at neutral pH, e.g. Urea (H2NCONH2) is used in denaturing PAGE; Formamide (HCONH2) is used Southern and Northern blotting. Mechanism Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (stacking interaction) is reduced Denaturation of the strands

Viscosity（粘性） Consequence of the DNA high viscosity Molecular Biology Viscosity（粘性） Consequence of the DNA high viscosity A high axial ratio (2 nm in diameter, and a length of micrometers, millimeters or even several centimeters in the Eukaryotic chromosomes) Relatively stiff Applications: Long DNA molecules can easily be damaged by shearing force, or by sonication. Pay attention to avoid shearing problem when need to isolate intact very large DNA molecule.

DNA and 8M CsCl has a similar density, around 1.7 g cm-3 Molecular Biology Buoyant density DNA and 8M CsCl has a similar density, around 1.7 g cm-3 Purifications of DNA: equilibrium density gradient centrifugation ΡDNA=1.66+0.098%(G+C) Protein floats RNA pellets at the bottom

C3 Spectroscopic and Thermal Properties of Nucleic Acids Molecular Biology C3 Spectroscopic and Thermal Properties of Nucleic Acids

Molecular Biology UV absorption Nucleic acids absorb UV light due to the conjugated aromatic nature of the bases The wavelength of maximum absorption of light by both DNA and RNA is 260 nm (lmax = 260 nm) Applications: can be used for detection, quantitation and assessment of purity (A260/A280)

Quantitation of nucleic acids Molecular Biology Hypochromicity Caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. A260 value: dsDNA<ssDNA or RNA<nucleotide Quantitation of nucleic acids Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20; The corresponding value for ssDNA and RNA is approximately 25 The values for ssDNA and RNA are approximate for two reason: The values are the sum of absorbance contributed by the different bases (e : purines > pyrimidines) The absorbance values also depend on the amount of secondary structures in a given molecule.

Molecular Biology Purity of DNA The approximate purity of dsDNA preparations (see Topic G2) may be estimated by determination of the ratio of absorbance at 260 and 280 nm (A260/A280). A260/A280: dsDNA = 1.8 pure RNA = 2.0 Protein <1, 0.5 or so

Thermal denaturation/melting Molecular Biology Thermal denaturation/melting Heating also leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA. RNA: the absorbance increases gradually and irregularly DNA: the absorbance increases cooperatively. melting temperature (Tm): the temperature at the mid-point of the smooth transition, which has a 20% increase in absorbance. 80-100 °C for long DNA molecules

Note: Relative concepts Molecular Biology Renaturation Rapid cooling: allows only the formation of local region of base paring. The decreased in A260 is rather small. Slow cooling: allows time for whole complementation of dsDNA. Absorbance in 260nm decreases greatly and cooperatively. Note: Relative concepts 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)

Molecular Biology C4 DNA Supercoiling Closed-circular DNA: Almost all DNA molecules in cells can be considered as circular Supercoiling: Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding of the double helix. (Why?) Topoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.

Learn about several concepts: Molecular Biology Learn about several concepts: 1, Linking Number (Lk): the number of double-helical turns in the DNA molecule. 2. The level of supercoiling may be quantified in terms of the change in linking number (ΔLk) from that of the unconstrained (relaxed) closed-circular molecule (Lk°). 2, Twist（缠绕）:the local winding up or unwinding of the double helix 3, writhe（扭曲) : the coiling of the helix axis upon itself. Twist and writhe are interconvertible according to the equation ΔLk = ΔTw + ΔWr.

Molecular Biology A rubber tubing model for DNA supercoiling, which illustrates the change in DNA conformation. The changes in linking number are indicated (see text for details). Conformational flexibility in supercoiled DNA as modeled by rubber tubing. The changes in twist and writhe at constant linking number are shown (see text for details).

Molecular Biology 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.

Molecular Biology The mechanisms of (a) type I and (b) type II topoisomerases (see text for details).

Molecular Biology Intercalator Ethidium bromide locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe. (a) Ethidium bromide; (b) the process of intercalation, illustrating the lengthening and untwisting of the DNA helix.