Download - Nitrogenous bases
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Nitrogenous bases
Bicyclic purines
Monocyclic pyrimidine
Thymine (T) is 5-methyluracil (U)
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NucleosidesIn nucleic acids, the bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside
Glycosidic (glycoside, glycosylic) bond ( 糖苷键 )
R Ribose or 2’-deoxyribose
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C1 Nucleic Acid Structure
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.
Deoxynucleotides (deoxyribose containing)
Ribonucleotides (ribose containing)
Phosphate diester bonds
4
5 12
67
9 21
54
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C1 Nucleic Acid Structure
Phosphodiester bonds
DNA/RNA sequence:From 5’ end to 3’ endExample:5’-UCAGGCUA-3’= UCAGGCUA
3’ end: free hydroxyl (-OH) group
5’end: not always has attached phosphate groups
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C1 Nucleic Acid StructureDNA double helix
•Watson and Crick , 1953.•Two separate strands Antiparellel (5’3’ direction)Complementary (sequence)Base pairing: hydrogen bonding that holds two strands together
Essential for replicating DNA and transcribing RNA
• Sugar-phosphate backbones (negatively charged): outside• Planar bases (stack one above the other): inside
back
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Base pairing via hydrogen bonds
A:T G:C
1
2345
678
9
123
4 56
2
1
34
5 678
9
2 13
4 5
6
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•Double helix
•B form:
Right-handed
10 base pairs/turn
34 Å /turn
Diameter: ca. 20 Å
Other forms:
A: 11 bases/turn, base plate 20° slant
Z: 12 bases/turn, left-handed helical, one groove
C1 Nucleic Acid Structure
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C1 Nucleic Acid Structure
RNA Secondary StructureSingle stranded, no long helical structure like double-stranded DNAGlobular conformation with local regions of helical structure formed by intramolecular hydrogen bonding and base stacking.
tRNA(clover-like)
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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 acids
Protein Nucleic AcidsFibrous protein Globular protein Helical DNA Globular RNAStructural proteins Enzymes,
antibodies, receptors etc
Genetic information maintenance
Ribozome RNATransfer RNA (tRNA)Signal recognition
C1 Nucleic Acid Structure
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C1 Nucleic Acid Structure
Modified Nucleic Acids
Modifications correspond to numbers of specific roles. We will discuss them in some related topics. For example, methylation of A and C to avoid restriction digestion of endogenous DNA sequence (Topic G3).
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C2 Chemical and Physical Properties of Nucleic Acids
Stability of Nucleic Acids
1. Hydrogen bonding • Contribute to specificity, not overall stability of DNA helix• Stability lies in the stacking interactions between base pairs
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 (base stacking & hydrophobic effect). • This stacking is maximized in double-stranded DNA
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C2 Chemical and Physical Properties of Nucleic Acids
Effect of Acid & applications
Strong acid + high temperature completely hydrolyzed to (perchloric acid+100°C) bases, riboses/deoxyribose, and phosphate
pH 3-4 apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broke
n ]
Maxam and Gilbert chemical DNA sequencing:A DNA sequencing technique based on chemical removal and modificati
on of bases specifically and then cleaving the sugar-phosphate backbone of the DNA and RNA at particular bases (J2)
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C2 Chemical and Physical Properties of Nucleic Acids
Effect of Alkali & Application
DNA denaturation at high pH
keto form enolate formketo form enolate form
Base pairing is not stable anymore because of the change of tautomeric (异构 ) states of the bases, resulting in DNA denaturation
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RNA hydrolyzes at higher pH because of 2’-OH groups in RNA
Effect of Alkali & Application
RNA is unstable at higher pH
OH free 5’-OH
2’, 3’-cyclic phosphodiester
alkali
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Chemical DenaturationUrea (H2NCONH2) ( 尿素) : denaturing PAGE Formamide (HCONH2) (甲酰胺) and Formaldehyde ( 甲醛 ): Northern blot
Disrupting the hydrogen bonding of the bulk water solution
Hydrophobic effect (aromatic bases) is reduced
Denaturation of strands in double helical structure
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Viscosity
Reasons for the DNA high viscosity 1. High axial ratio2. Relatively stiff
Applications1. Long DNA molecules can easily be shortened by
shearing force.2. When isolating very large DNA molecule, always
avoid shearing problem
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Buoyant density1.7 g cm-3, a similar density to 8M CsCl. Rho=1.66+0.098 (GC)%Purifications of DNA: equilibrium density gradient centrifugation
RNA pellets at the bottom
Protein floats
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Spectroscopic and Thermal Properties of Nucleic Acids
1. 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 (max = 260 nm) - Applications: detection, quantitation, assessment of purity (A260/280)
2. Hypochromicity Fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide
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3. Quantitation of nucleic acids Extinction coefficient (): 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 ( : purines > pyrimidines) - The absorbance values also depend on the amount of secondary structures due to hypochromicity.
4. Purity of DNA A260/280: dsDNA--1.8 pure RNA--2.0 protein--0.5
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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 cooperativelyMelting temperature (Tm) the temperature at which 40% increase in
absorbance is achieved.
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6. Renaturation:
Rapid cooling Only allow the formation of local base paring Absorbance is slightly decreasedSlow 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)
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DNA Supercoiling1. Almost all DNA molecules in cells are on average negatively
supercoiled.
2. Supercoiled DNA has a higher energy than relaxed DNA. Ne
gative supercoiling may thus facilitate cellular processes whi
ch require the unwinding of the helix, such as transcription i
nitiation or replication
3. Topoisomerases exist in cell the regulate the level of supercoi
ling of DNA molecules. (important to know in the sense of gen
e expression)
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Linker number a topological property of a closed-circular DNA, which can be changed only if one or both of the DNA backbones are broken.
Topoisomer A molecule of a given linking number is known as a topoisomer. Topoisomers of the same molecule differ from each other only in their linker number.
The conformation (geometry) of the DNA can be altered while the linking number remains constant. Writhe (wrap around,缠绕 ) and Twist (扭转 ) changes are to measure the conformational change of a DNA molecule (Lk = Tw + Wr).
1. The topological change (Lk) in supercoiling of a DNA molecule is partitioned into a conformational change of twist (Tw )and/or a change of writhe (Wr).
2. For a given isomer of a circular closed DNA (Lk = 0), the increase in twist will cause a corresponding decrease in writhe.
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Ethidium bromide (intercalator 插入物 ) locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.
Topoisomerases
Type I break one strand of the DNA (via P-tyrosine bond) , 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. (ATP)
Bacterial gyrase ( 旋转酶 ) introduce negative supercoiling. ATP.
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Summary:
1. Nucleic acid structure
bases > nucleosides (base+sugar) > nucleotides (nucleoside+phosphate) > polynucleotides /DNA/RNA (via 3’,5’-phosphodiester bond) > DNA double helix/RNA secondary structure
2. Chemical and physical properties
stability support, effect of acid and alkali, chemical denaturation, viscosity, buoyant density
3. Spectroscopic and thermal properties
4. DNA supercoiling: Linking number (twist and writhe))