3 The diffraction pattern from the DNA optical transform slide shows the central cross pattern indicative of the helical arrangement of the strands of DNA. It also shows a missing 4th layer line which is the result of the two strands of the double helix being offset by 3/8 of a period. The strongly diffracting phosphorus atoms create the diamonds. The satelites above and below the cross are attributed to the base pairs.
4 Molecular Biology Course C1 Nucleic Acid Structure (DNA & RNA): basesnucleosides nucleotide phosphodiester bonds primary sequence structure, modified nucleic acidsC2 Chemical and Physical Properties of Nucleic Acids (DNA & RNA): stability (force), chemical properties (acid, alkali, chemical denaturation), physical properties (viscosity, buoyant density)C3 Spectroscopic and Thermal Properties of Nucleic Acids (DNA & RNA): UV absorption, hyperchromocity, quantitation and purityC4 DNA Supercoiling (DNA): closed circular molecule, supercoiling & energy, topoisomer & topoisomerase
5 Bicyclic Purines: Monocyclic pyrimidine: C. Properties of nucleic acidsC1 Nucleic Acid Structure (DNA & RNA): BasesBicyclic Purines:Monocyclicpyrimidine:Thymine (T) is a 5-methyluracil (U)
6 Adenosine, guanosine, cytidine, thymidine, uridine C. Properties of nucleic acidsC1 Nucleic Acid Structure (DNA & RNA): NucleosidesThe bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleosideGlycosidic (glycoside, glycosylic) bond(糖苷键)RRibose or2’-deoxyriboseAdenosine, guanosine, cytidine, thymidine, uridine
7 (containing deoxyribose) C. Properties of nucleic acidsC1 Nucleic Acid Structure (DNA & RNA): NucleotidesA 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 bondsDeoxynucleotides(containing deoxyribose)Ribonucleotides(containing ribose)
9 5’end: not always has attached phosphate groups 3’ end: free hydroxyl C. Properties of nucleic acidsC1 Nucleic Acid Structure (DNA & RNA): Phosphodiester bonds & primary sequencePrimary sequence:5’end: not always has attached phosphate groups3’ end: free hydroxyl(-OH) groupPhosphodiester bond
10 The genetic material of all organisms except for some viruses C. Properties of nucleic acidsC1 Nucleic Acid Structure : DNA double helixWatson and Crick , 1953The genetic material of all organisms except for some virusesThe foundation of the molecular biology
11 C. Properties of nucleic acids Essential for replicating DNA and transcribing RNATwo separate strands Antiparellel (5’3’ direction)Complementary (sequence)Base pairing: hydrogen bonding that holds two strands together3’5’Sugar-phosphate backbones (negatively charged): outsidePlanner bases (stack one above the other): inside3’5’back
12 A:T G:C Base pairing C. Properties of nucleic acids 4 1 3 2 6 7 5 8 1 9423A:TG:CBase pairingback
13 Helical turn: 10 base pairs/turn 34 Ao/turn C. Properties of nucleic acidsHelical turn:10 base pairs/turn34 Ao/turnback
14 C1 Nucleic Acid Structure : RNA structure C. Properties of nucleic acidsC1 Nucleic Acid Structure : RNA structureSingle stranded nucleic acidSecondary structure are formed some timeGlobular tertiary structure are important for many functional RNAs, such as tRNA, rRNA and ribozyme RNAForces for secondary and tertiary structure: intramolecular hydrogen bonding and base stacking.
16 C. Properties of nucleic acids Conformational 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 acidsProteinNucleic AcidsFibrous proteinGlobular proteinHelical DNAGlobular RNAStructural proteinsEnzymes,antibodies,receptors etcGenetic information maintenanceRibozymesTransfer RNA (tRNA)Signal recognition etc.
17 C. Properties of nucleic acids C1 Nucleic Acid Structure : Modified Nucleic AcidsModifications 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 (Topic G3).
18 C2 Chemical and Physical Properties of Nucleic Acids Molecular Biology CourseC2 Chemical and Physical Properties of Nucleic AcidsStability of Nucleic AcidsEffect of Acid & applicationsEffect of alkali & applicationsChemical denaturationViscosity & applicationsBuorant density & applicationChemical propertiesPhysical propertiesback
19 Stability of Nucleic Acids C. Properties of nucleic acidsStability of Nucleic AcidsHydrogen bondingDoes not normally contribute the stability of nucleic acids or proteinContributes to specific structures of these macromolecules. For example, a-helix, b-sheet, DNA double helix, RNA secondary structure2. 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 otherThis stacking is maximized in double-stranded DNAFig
20 C. Properties of nucleic acids Effect of AcidStrong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphatepH 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
21 Effect of Alkali & Application C. Properties of nucleic acidsEffect of Alkali & ApplicationHigh pH (> 7-8) has subtle (small) effects on DNA structureHigh pH changes the tautomeric (互变异构)state of the basesenolate formenolate formketo formketo formBase pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation
22 RNA hydrolyzes at higher pH because of 2’-OH groups in RNA C. Properties of nucleic acidsRNA hydrolyzes at higher pH because of 2’-OH groups in RNA2’, 3’-cyclic phosphodiesteralkaliOHfree 5’-OHRNA is unstable at higher pH
23 Disrupting the hydrogen bonding of the bulk water solution C. Properties of nucleic acidsChemical DenaturationUrea (H2NCONH2) (尿素）: denaturing PAGEFormamide (HCONH2) （甲酰胺）: Northern blotDisrupting the hydrogen bonding of the bulk water solutionHydrophobic effect (aromatic bases) is reducedDenaturation of strands in double helical structure
24 Viscosity（粘性） Reasons for the DNA high viscosity Applications: C. Properties of nucleic acidsViscosity（粘性）Reasons for the DNA high viscosityHigh axial ratioRelatively stiffApplications:Long DNA molecules can easily be shortened by shearing force. Remember to avoid shearing problem when isolating very large DNA molecule.
25 Buoyant density (DNA) 1.7 g cm-3, a similar density to 8M CsCl C. Properties of nucleic acidsBuoyant density (DNA)1.7 g cm-3, a similar density to 8M CsClPurifications of DNA: equilibrium density gradient centrifugationProtein floatsRNA pellets at the bottomback
26 C3 Spectroscopic and Thermal Properties of Nucleic Acids Molecular Biology CourseC3 Spectroscopic and Thermal Properties of Nucleic AcidsUV absorption:nucleic acids absorb UV light due to the aromatic basesThe wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm)Applications: detection, quantitation, assessment of purity (A260/A280)2. 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
27 3. Quantitation of nucleic acids C. Properties of nucleic acids3. Quantitation of nucleic acidsExtinction coefficients: 1 mg/ml dsDNA has an A260 of 20ssDNA and RNA, 25The values for ssDNA and RNA are approximateThe 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 due to hypochromicity.Purity of DNAA260/A280:dsDNA--1.8pure RNA--2.0protein--0.5
28 C. Properties of nucleic acids 5. Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.RNA: the absorbance increases gradually and irregularlyDNA: the absorbance increases cooperatively.melting temperature (Tm): the temperature at which 40% increase in absorbance is achieved.back
29 6. Renaturation: C. Properties of nucleic acids Rapid cooling: only allow the formation of local base paring Absorbance is slightly decreasedSlow cooling: whole complementation of dsDNA. Absorbance decreases greatly and cooperatively.Fig. 2.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)
30 DNA Supercoiling Closed circular molecule Supercoiling & energy C. Properties of nucleic acidsDNA SupercoilingClosed circular moleculeSupercoiling & energyTopoisomer & topoisomerase
31 C. Properties of nucleic acids Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled.Counter helical turn
32 C. Properties of nucleic acids 2. Negative supercoiled DNA has a higher torsional (扭转的) energy than relaxed DNA, which facilitates the unwinding of the helix, such as during transcription initiation or replicationTopoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.
33 C. Properties of nucleic acids 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.
34 C. Properties of nucleic acids Ethidium bromide (intercalator): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.TopoisomerasesType I: break one strand of the DNA , and change the linking number in steps of ±1.Type II: break both strands of the DNA , and change the linking number in steps of ±2.