Chapter 7. Nucleic Acids (核酸)
I.
Introduction: Nucleotides and nucleic acids
(核苷酸与核酸)
II. Nucleic acid structure (核酸的结构)
III. Physical and chemical properties of nucleic
acids (核酸的物理化学性质)
1
I. Introduction: Nucleotides and Nucleic Acids
(核苷酸与核酸简介)
1. History of the nucleic acid research (核酸研究的历史)
2. Functions of nucleic acids (核酸的作用)
3. Compositions of nucleic acids (核酸的组成)
DNA and RNA are
molecular repositories of
genetic information.
2
1. History of the nucleic acid research (核酸研究的历史)
When?
Who?
What?
1868
F. Miescher
Discovery of “nuclein” (核素)
1889
R. Altmann
First use of the name “nucleic acid”
1940s
Chargaff
Chargaff’s rules
1944
O.T. Avery et al.
DNA is the bearer of genetic infomation
1952*
Watson & Crick
DNA double helix model (DNA双螺旋模型)
1958
F. Crick
“Central dogma” (中心法则)
1973
Cohen & Boyer
Recombinant DNA technique (DNA重组技术)
1975*
F. Sanger
DNA sequencing (DNA测序)
1981*
T. Cech
Ribozyme (核酶)
1983*
K.B. Mullis
PCR (聚合酶链反应)
1990-2003
R. Dulbecco
Human Genome Project (人类基因组计划)
1998*
A. Fire & C. Mello
RNA interference (RNA干扰机制)
3
Nobel Laureates
in DNA
4
Nobel Prizes in DNA
Laureates
Prize
Contribution
1959
Ochoa, Kornberg
Physiology/
Medicine
“for their discovery of the mechanisms in the biological synthesis of
ribonucleic acid and deoxyribonucleic acid”
1962
Watson, Crick,
Wilkins
Chemistry
"for their discoveries concerning the molecular structure of nucleic acids and
its significance for information transfer in living material"
1968
Holley, Khorana,
Nirenberg
Physiology/
Medicine
“for their interpretation of the genetic code and its function in protein synthesis”
1978
Arbers, Nathans,
Smith
Physiology/
Medicine
“for the discovery of restriction enzymes and their application to problems of
molecular genetics”
1980
Berg, Gilbert,
Sanger
Chemistry
"for his fundamental studies of the biochemistry of nucleic acids, with
particular regard to recombinant-DNA", "for their contributions concerning the
determination of base sequences in nucleic acids"
1989
Altman, Cech
Chemistry
"for their discovery of catalytic properties of RNA"
1993
Mullis, Smith
Chemistry
"for contributions to the developments of methods within DNA-based
chemistry"
1997
Boyer, Walker
Chemistry
"for their elucidation of the enzymatic mechanism underlying the synthesis of
adenosine triphosphate (ATP)"
2002
Brenner, Horvitz,
Sulston
Physiology/
Medicine
"for their discoveries concerning 'genetic regulation of organ development and
programmed cell death'"
2006
Roger Kornberg
Chemistry
"for studies of the molecular basis of eukaryotic transcription"
5
复制
转录
DNA replicates its
information.
DNA codes for the
production of mRNA.
mRNA is processed and
migrates from the nucleus to
the cytoplasm.
翻译
mRNA carries coded
information to ribosomes
(with rRNAs the stuctural
components), where the
info is translated by tRNA
into a specific sequence of
amino acids.
6
2. Functions of nucleic acids (核酸的作用)
DNA
Nucleic acids
(核酸)
RNA
Storage and transmission of biological
information (储存和传递生物信息)
Ribosomal RNA
rRNA
(核糖体RNA)
Structural components of
ribosomes (核糖体的结构成分)
Messenger RNA
mRNA
(信使RNA)
Carrying genetic information from
genes to ribosomes (把遗传信息从
基因运到核糖体)
Transfer RNA
tRNA
(转移RNA)
Translating the information in
mRNA into a specific sequence of
amino acids (把mRNA的信息转译
成氨基酸序列)
Other RNAs
7
DNA stores genetic information
The AveryMacLeodMcCarty
experiment
(1944)
The
HersheyChase
experiment
(1952)
8
3. Compositions of nucleic acids (核酸的组成)
Base (碱基)
Nucleic acid
(核酸)
Nucleotide
(核苷酸)
Nucleoside
(核苷)
Pentose (戊糖)
Phosphate
(磷酸)
I. Introduction
9
A Nucleotide
Adenosine Mono Phosphate (AMP)
Phosphate
HO
H+
Nucleotide
(核苷酸)
OH
P
Base
O
N
OH
O
OH
N
5’CH2
4’
O
1’
Sugar
3’
OH
NH2
2’
OH
H
N
N
Nucleoside
(核苷)
10
RNA
DNA
I. Introduction
11
1). Nitrogenous bases
(碱基)
嘧啶
Purine bases – A, G
嘌呤碱
嘌呤
G
A
DNA
Pyrimidine bases – T, C
嘧啶碱
腺嘌呤
Purine bases – A, G
嘌呤碱
C
鸟嘌呤
胸腺嘧啶
U
T
RNA
Pyrimidine bases – U, C
嘧啶碱
胞嘧啶
12
尿嘧啶
Minor bases (稀有碱基)
Minor bases of DNA
Minor bases of tRNA
13
2). Pentoses (戊糖)
RNA
DNA
D-ribose (D-核糖)
2’-deoxy-D-ribose (D-2’-脱氧核糖)
5’
4’
1’
3’





2’
Difference between DNA and RNA: C-2’
D-pentose (D型戊糖)
-configuration (-构型)
Furanose ring form (呋喃糖环状结构)
The pentose ring is not planar but in puckered conformations
14
(戊糖环不是完全平面).
Puckered Conformations of ribofuranose rings in nucleotides
(核苷酸内呋喃糖环的四种折叠构象)
exo (外式)
endo (内式)
15
3). Nucleosides (核苷)
7
8
HO
HO
H
H
OH
H
OH
Syn (顺式)

4’H3’
H
OH
5
4
4
6
N
H
H
2’
OH
Anti (反式)
1
6
2
1’-N9-糖苷键
N3
5
N1
3
5’
O
O
9
N
N
N


N
N
N
H
NH2
NH2
NH2
N
HO
5’
H
OH
O
-N1-糖苷键
O
4’H
3’
2
H
1’
H
2’
H
Anti (反式)
Adenosine
Deoxycytidine
(腺嘌呤核苷)
(胞嘧啶脱氧核苷)
A nucleoside (核苷) = a pentose (戊糖) + a base (碱基)
N-glycosidic bond (糖苷键):
 C-1’ (pentose) – N-1 (pyrimidine,嘧啶碱)
 C-1’ (pentose) – N-9 (purine,嘌呤碱)
Conformation (构象):
 Furanose and base rings are perpendicular to each other (戊糖和碱基相互垂直)
 Syn (顺式), anti (反式): Pyrimidine nucleosides favor the anti, while purine
16
nucleosides can adopt either syn or anti.
4). Nucleotides (核苷酸)
NH2
磷酸酯键
NH2
N
N
N
糖苷键
O
-
O
P
N
N
O
N
CH2OH
O
O
H
H
O
H
O
H
O-
H
H
H
H
OH
OH
-
O
Adenosine 5’-monophosphate
(5’-腺嘌呤核苷酸)
AMP
H
P
O-
O
Deoxycytidine 3’monophosphate
(3’-胞嘧啶脱氧核苷酸)
3’-dCMP
 A nucleotide is a phosphoester of a nucleoside. (核苷酸是核苷的磷酸酯)
 -OH groups on the ribose available for esterification to form a nucleotide (戊糖
上能与磷酸酯化形成核苷酸的羟基):
 Ribose ring (核糖环): C-2’, C-3’, C-5’
 Deoxyribose ring (脱氧核糖环): C-3’, C-5’
 Acidic properties: pKa1 = 1.0, pKa2 = 6.0
17
Some adenosine monophosphates
(单磷酸腺苷)
5’-AMP
2’-AMP
3’-AMP
2’,3’-cAMP
18
Deoxyribonucleotides
(脱氧核糖核苷酸)
腺嘌呤脱氧核苷酸
19
Ribonucleotides
(核糖核苷酸)
腺嘌呤核苷酸
20
O
Cyclic nucleotides
-- Important regulators of cellular metabolism
NH
N
H2
5’
C
N
O
O
O-
H
H
3’
OH
H
O
P
N
NH2
3’,5’-Cyclic GMP
(cGMP)
H
O
Nucleoside diphosphates and triphosphates (NDPs and NTPs)
NH2
N
N
O
-
O P O
O
NH2
N
N
N
O
-
H
O
Pi
H
H
OH
H
OH
N
O
-
O P O P O
O
-
O
-
O
H
Adenosine 5’-monophosphate
(AMP)
Pi
H
H
OH
H
OH
-
O

O-
N
O
O
O-
 
H
Adenosine 5’-diphosphate
(ADP)
N
O P O P O P O
O-
N
NH2
Pi
O
N
H
H
OH
H
OH
N
N
Adenosine 5’-triphosphate
(ATP)
21
5). Nucleic acid (核酸)


Nucleic acids are linear polymers of nucleotides linked by
3’,5’-phosphodiester bridges. (核酸是由核苷酸通过
3’,5’-磷酸二酯键连接而成的链状聚合物)
Two major classes: DNA and RNA
 DNA (deoxyribonucleic acid, 脱氧核糖核酸)
– Polymer of deoxyribonucleotides
 RNA (ribonucleic acid,核糖核酸)
– Polymer of ribonucleotides
I. Introduction

Chemical differences between DNA and RNA:
 DNA contains 2’-deoxyribose instead of ribose
(DNA含脱氧核糖而不是核糖)
 DNA contains thymine instead of uracil.
(DNA含胸腺嘧啶而不是尿嘧啶)

Greater stability of DNA over RNA
22
Phosphodiester linkage (磷酸二酯键)
O
-
O
O
P
O
-
O
P
O
O
CH2
H
H
CH2
B
O
H
3’
OH
OH
H
H
H
H
-H2O
-
-
O
O
P
O
O
H
3’
O
H
P
I. Introduction
5’ CH2
H
O
B
O
5’ CH2
H
H
H
H
H
3’,5’phosphodiester bond
(磷酸二酯键)
O
O
O
H
B
O
B
O
H
H
O
H
H
For DNA, only the 3’- and 5’-OH groups are available for
internucleotide phosphodiester bonds.
23
II. Nucleic Acid Structure
1. Overview of the nucleic acid structure (核酸结
构总述)
2. Primary structure of nucleic acids (核酸的一级
结构)
3. Secondary structure of DNA (DNA的二级结构)
4. Tertiary structure of DNA (DNA的三级结构)
5. Secondary and tertiary structure of RNA (RNA
的二级和三级结构)
24
1. Overview of the nucleic acid
structure (核酸结构总述)
1)
2)
II. Structures
3)
Primary structure (一级结构) – Covalent structure and
nucleotide sequence (共价结构和核苷酸序列)
Secondary structure (二级结构) – regular, stable
structure taken up by some or all of the nucleotides in a
nucleic acid (核酸内全部或部分核苷酸形成的一些规
则的稳定的结构)
Tertiary structure (三级结构) – complex folding of large
chromosomes (染色体) within eukaryotic chromatin (染
色质) and bacterial nucleoids (大染色体的复杂折叠状
态)
25
2. Primary structure of nucleic acids
P.482
(核酸的一级结构)
1)
2)
3)
II. Structures
4)
5)
6)
Phosphodiester bonds link successive nucleotides in
nucleic acids (核酸中的核苷酸以磷酸二酯键彼此相连)
The covalent backbones of nucleic acids consist of
alternating phosphate and pentose residues. (核酸的共
价主链由磷酸根和戊糖残基交替出现组成)
The backbones for both DNA and RNA are hydrophilic.
(两种核酸的主链均亲水)
The phosphate groups are completely ionized and
negatively charged at pH 7. (中性条件下磷酸基团彻底
电离并带负电)
Both DNA and RNA have unbranched linear or cyclic
structures. (两种核酸均成直链或环状结构，没有支链)
The bases serve as “Information Symbols”.
26
Presentations (表示方式)
T
1’
1’
C
1’ 1’
3’
3’
3’
3’
3’
5’ 5’
5’
5’
5’
1’
pA-T-G-C-AOH
pApTpGpCpA
pATGCA
5’3’走向：上下，左右
27
3. Secondary structure of DNA
(DNA的二级结构)
II. Structures
1) Chargaff’s rules (Chargaff规则)
2) Watson & Crick’s double helix model (DNA分
子双螺旋结构模型)
3) Structural Variation in DNA (DNA的结构变化)
4) Different helix forms of DNA (DNA的几种螺
旋类型)
5) Unusual structures of certain DNA sequences
(某些DNA序列采取的不寻常结构)
28
1). Chargaff’s rules (Chargaff规则)



II. Structures

The base composition of DNA generally varies from one
species to another. (不同物种的DNA碱基组成不一样)
DNA specimens isolated from different tissues of the same
species have the same base composition. (同一物种不同
组织和器官的DNA碱基组成是一样的)
The base composition of DNA in a given species does not
change with an organism’s age, nutritional state, or
changing environment. (同一物种的DNA碱基组成不受
生长发育、营养状况、环境条件的影响)
In all cellular DNAs, regardless of the species,
A = T, G = C,
A+G = T+C (嘌呤碱 = 嘧啶碱)
29
Major
groove
G
A T
-
-
A T
C G
G C
0.34 nm
T A
-
-
Crick:“We have discovered
the secret of life!”
3.4 nm
1 nm
-
-
-
- G TC A
- C
-
-
-
-
-
-
-
-
G
-
-
Minor
groove
C
G C
T A
A T
-
2) Watson & Crick’s double helix model
(DNA分子双螺旋结构模型)
-
-
-
-
30
Watson & Crick’s double helix model
(DNA分子双螺旋结构模型)
5’
5’
3’
3’
Watson and Crick’s model was postulated based on:
 Chargaff’s rule: A = T, G = C, A+G = T+C
 Franklin and Wilkins’ X-ray diffraction studies on
the structure of DNA fibers: helical, 3.4 nm
Watson & Crick’s double helix model:
 Two helical DNA chains wind around the same
axis to form a right-handed double helix. (两条
DNA链绕同一中心轴成右手双螺旋)
 The two DNA strands:
Held by H-bonds formed between specific
base pairs (两条DNA链靠彼此碱基间所成
氢键结合)
Complementary (两条DNA链互补)
Antiparallel (两条DNA链反向平行)
 3.4 nm/10 base pairs for each complete turn (3.6
nm/10.5 pairs in aqueous solution) (每旋转一周
有10个核苷酸，螺距3.4 nm)
31
5’Phosphate group
NH2
O
N
O
O
O
O
N
O
CH2
H
NH2
O
H
H2O
N
CH2
N
O
O
C≡ G
O
H
O
OH
CH2
P
HO
O
3’Hydroxyl group
HO
P
O
P
NH2
N
G≡ C
O
H2O
NH
N
O
OH
H
O
CH2
HO
P
O
H
P
CH2
O
H
O
HO
N
A=T
O
HO
N
N
O
OH
5’Phosphate
group
HO
CH2
H
O
P
HO
O
D
N
A
3’Hydroxyl group
OH
32
Watson & Crick’s double helix model
(DNA分子双螺旋结构模型)
C-2’ endo conformation (内式)
Parallel to the axis (与纵轴平行)
Deoxyribose (脱氧核糖)
Outside
Phosphate group (磷酸基团)
Bases (碱基)
Inside
Perpendicular to the axis (与纵轴垂直)
33
Two forces to hold the DNA double helix
(形成双螺旋结构的两个重要作用力)
Base-stacking interactions
A=T
(碱基堆积)
 Nonspecific (非专一性)
 Hydrophobic stacking interaction
 Major contribution to the stability
of the double helix (使双螺旋结
构稳定)
H-bonding between base pairs
GC
(氢键)
 Specific (专一性)
 A=T, G  C
 Major contribution to the
complementarity between DNA
strands (使双螺旋结构互补)
34
Watson & Crick’s double helix model
(DNA分子双螺旋结构模型)
II. Structures
Mechanism for the transmission
of genetic information (遗传信息
的传递机理):
a) Separating the two strands
(两股DNA单链分开)
b) Synthesizing a
complementary strand for
each (每股DNA单链作为母
链各合成出一条与之互补
的子链)
35
4) Different helix forms of DNA (DNA的几种螺旋类型)
DNA structure varies upon the change in environmental conditions.
P.488
Watson-Crick structure
Stable under physiological conditions

Differences:





II. Structures
Helical sense
Diameter
Base pairs per helical turn
Helix rise per base pair
Base tilt normal to the
helix axis
 Sugar pucker
conformation
 Glycosyl bond
conformation

Interchangeable upon the
change in:




Crystal structures
Humidity
Salt concentration
Ion types
Organic solvents
36
Comparison of A, B, and Z forms of DNA
37
5) Unusual structures of certain DNA sequences
(某些DNA序列采取的不寻常结构)

Hairpins and cruciforms (发夹型和十字型)
 Triplex and Tetraplex (三股螺旋和四股螺旋)
 H-DNA
II. Structures
38
Hairpins and cruciforms
(发夹型和十字型)
回文
发夹
镜像重复
十字型
39
P.489
Triplex and Tetraplex
(三股螺旋和四股螺旋)
Hoogsteen pairing (Hoogsteen配对) – Non-Watson-Crick pairing
一些核苷酸的碱基在形成Watson-Crick式碱基配对之后还能与其它核
苷酸的碱基以额外的氢键配对。这种非Watson-Crick式的碱基配对叫
做Hoogsteen配对。
Triplex (三股螺旋的DNA)
T=A*T
C  G * C+
T=A*A
CG*G
 Most stable at low pH
 Readily formed within long
sequences containing only purines
or only pyrimidines in a given
strand
Tetraplex (四股螺旋的DNA)
G
G
G
G
 Only occuring for DNA
sequences with a very high G
proportion
 Stable over a wide range of
conditions
40
Watson-Crick pairing
Hoogsteen pairing
Triplexes are most stable at low pH
pKa for C in triplex > 7.5
pKa for C in free form = 4.2
41
H-DNA
H-DNA is usually formed when a sequence of polypyrimidine or polypurine
P.490




nucleotides incorporates a mirror repeat. (当DNA的一段多聚嘧啶核苷酸或多聚嘌呤核
苷酸组成镜像重复，即可回折产生H-DNA)
Protonation of C is required for Hoogsteen pairing with G. (C需要质子化)
B-DNA  H-DNA under acidic conditions (酸性条件下发生BH-DNA的转变)
Design of a synthetic DNA strand to form a triplex DNA, thus disrupting gene
expression – potential application in medicine and agriculture
42
5. Secondary and tertiary structure of RNA
(RNA的二级和三级结构)
Bases

RNAs are typically single-stranded (单链RNA)
 Right-handed helical conformation (右手螺旋)
 Stabilized by base-stacking interactions: purine-
Phosphates
purine>purine-pyrimidine > pyrimidine-pyrimidine
 Pairing with RNA or DNA (与RNA或DNA配对)

RNA-RNA or RNA-DNA duplexes (双螺旋)





Ribose
II. Structures

The paired strands are antiparallel (两股单链反向平行)
A-form is predominant (A形螺旋占优势)
B-form is impossible due to the 2’-OH
Base-stacking interactions (碱基堆积使结构稳定)
Base pairing patterns (碱基配对方式):
 G  C, A = U — Same as in DNA
 G = U — Different from DNA
 Unusual H bonds (不寻常的氢键)
RNA has no simple, regular secondary
structure -- Hairpins are the most common type
43
(发夹形是最常见的二级结构)
Hairpins are the most common type of secondary
structure in RNA (发夹形是RNA最常见的二级结构)
II. Structures
G=U
G=U
44
M1 RNA component of RNase P of E.coli
P.496
Secondary structure of tRNA
(tRNA的二级结构)
氨基酸臂
A cloverleaf
structure of
tRNA
反密码环
 Amino acid arm – forming the
aminoacyl-tRNA derivative
 D loop – containing D (二氢尿
嘧啶核苷)
 Anticodon loop – containing
the anticodon that recognizes
and base pairs with a particular
mRNA codon
 Extra loop – variable in size
 TC loop – recognition site for
ribosomes to bind tRNAs
D -- dihydrouridine, 二氢尿嘧啶核苷
 -- pseudouridine, 假尿嘧啶核苷
45
P.497
tRNA tertiary structure
(tRNA的三级结构)
Amino acid arm, -CCA
3’-OH end
 Stable
L-shaped tertiary form
 Major stabilizing factors:
 H-bonding between
bases in the D loop and
bases in the variable and
TC loops.
 Hydrophobic stacking
interactions
Anticodon loop
Three-dimensional structure
of yeast phenylalanine tRNA
46
P.502
III. Physical and chemical properties of
nucleic acids (核酸的物理化学性质)
1.
2.
3.
4.
Hydrolysis (水解)
Acid-base properties (酸碱性质)
Spectrophotometric properties (光谱性质)
Denaturation, renaturation, and hybridization
(变性，复性，杂交)
5. Nonenzymatic transformations (非酶催化的
转化)
47
P.502
1. Hydrolysis (水解)
Bonds subjected to
hydrolysis (可被水解的键):
 N-glycosyl bond (糖苷键)
 Phosphoester bond (磷酸
酯键)
Phosphoester bond
(磷酸酯键)
Hydrolysis by:
 Acid (酸水解)
 Base (碱水解)
 Enzyme (酶水解)
N-glycosyl bond
(糖苷键)
III. Properties
48
1. Hydrolysis (cont’d)
1) Acidic hydrolysis (酸水解)



The glycosyl bond is easier to be hydrolyzed than the
phosphoester bond. (糖苷键比磷酸酯键更易被水解)
Glycosyl bonds with purine bases are more unstable than those
with pyrimidine bases (嘌呤碱的糖苷键比嘧啶碱的糖苷键更
不稳定)
Glycosyl bonds between deoxyribose and purine bases are most
unstable (嘌呤碱与脱氧核糖之间的糖苷键对酸最不稳定)
2) Alkaline hydrolysis (碱水解)

III. Properties


The glycosyl bond is stable and cannot be hydrolyzed. (糖苷键
在碱性条件下不水解)
The phosphoester bond of RNA (but not DNA) can be
hydrolyzed. (RNA的磷酸酯键被水解，DNA的磷酸酯键不能)
DNA is stable under alkaline conditions. (DNA对碱稳定)
3) Enzymatic hydrolysis (酶水解)
49
Comparison of acidic and alkaline
hydrolysis of nucleic acids
Acidic hydrolysis
(酸水解)
Bonds to be
Glycosyl bond >
hydrolyzed
phosphoester bond
(可水解的共价键) (糖苷键 > 磷酸酯键)
Pentose
DNA > RNA
Base
(碱基)
Purine > pyrimidine
(嘌呤碱 > 嘧啶碱)
Alkaline
hydrolysis
(碱水解)
Phosphoester
bond only
(磷酸酯键)
RNA only
50
DNA Depurination upon acid hydrolysis
(DNA 脱嘌呤碱)
pH 1.6, 37oC
III. Properties
DNA
Apurinic acid
(无嘌呤酸)
51
Hydrolysis of RNA under Alkaline Conditions
2’,3’-环磷酸酯
2’-磷酸核苷酸 3’-磷酸核苷酸
52
P.503
Enzymes that hydrolyze nucleic acids
(水解核酸的酶)
Ribonuclease, RNase
(核糖核酸酶)
Substrate specificity
(底物专一性)
Deoxyribonuclease, DNase
(脱氧核糖核酸酶)
N-Glycosidase (N-糖苷酶)
Nuclease
(核酸酶)
Cleavage pattern
(对底物作用方式)
Endonuclease (核酸内切酶)
Exonuclease (核酸外切酶)
III. Properties
Phosphoester bond cleavage
(磷酸酯键的断裂)
(1) 3’-OH -- P
(2) 5’-OH -- P
(1)
(2)
53
P.504
2. Acid-base properties (酸碱性质)
III. Properties
1) H+ available for ionization (可电离的氢离子)
2) Amphipathy (两亲性)
3) Dissociation of the base, the nucleoside, and
the nucleotide (碱基、核苷、核苷酸的解
离): P.504-506
4) Titration curves (滴定曲线): P.506-507
54
胞嘧啶核苷酸
pK1’ = 0.7~1.6
pK3’ = 5.9~6.5
pK2’ = 4.15 (A, 腺嘌呤)
= 3.2 (G, 鸟嘌呤)
= 4.6 (C, 胞嘧啶)
= 9.5 (U, 尿嘧呤)
= 9.9 (T, 胸腺嘧啶)
NH2
OH
N+H
HO
P
O
pKa
O
N
CH2
H
O
H
H
H
OH
OH
pK4’ > 12
O
Free base Nucleoside
(自由碱基)
(核苷)
A
4.15
3.5
G
3.2
1.6
C
4.6
4.15
U
9.5
9.2
T
9.9
9.8
Nucleotides are amphipathic (核苷酸是两性电解质)
55
Ionization of Cytidine 5’-monophosphate
(胞嘧啶核苷酸的电离)
A
B
NH2
OH
HO-P=O
O
CH2 N
O
C
NH2
OHO-P=O
N+H
O
-H+
O
CH2 N
pK1’=0.8
HO OH
HO-P=O
O
CH2 N
O
-H+
O
NH2
O-
N +H
D
HO OH
-O-P=O
N
N
O
CH2 N
O
-H+
O
pK2’=4.5
NH2
O-
O
O
pK3’=6.3
HO OH
HO OH
Amphipathic ion (兼性离子)
pI =
pK1’ + pK2’
2
（等电点）
56
P.507
3. Spectrophotometric properties
(光谱性质)
1) Nucleic acids have strong absorption at ~260 nm because of
the conjugated double bonds in the base groups (由于碱基的共轭
双键，核酸在260 nm附近有强吸收)
2) DNA/RNA can be characterized by A260 (核酸可用A260鉴定)
3) Purity of a nucleic acid sample can be estimated by A260/A280
(核酸样品的的纯度可用A260/A280鉴定)
57
P.508
4. Denaturation, renaturation, and
hybridization (变性，复性，杂交)
 Denaturation
(变性) results in
disruption of the H bonds
between two paired strands. No
covalent bonds are broken. (核酸
变性导致两条单链之间的氢键
破裂，不涉及共价键的破裂)
 Complete
denaturation
 Partial denaturation
 Renaturation (复性): The
denatured DNA/RNA can be
renatured if the denaturing
conditions are removed. (除去变
性条件可使已变性的核酸复性)
– if partially denatured
 Slow – if completely denatured
 Fast
58
Denaturing conditions (变性条件)
pH extremes
– 酸碱变性
– Alkali is the preferred
denaturant.
 Strong H-bonding
solutes as denaturants
(变性剂)
– e.g. urea (尿素)
 Temperature
– 热变性
 Ionic strength
– DNA double helix is
more stable at a high
ionic strength.

pH > 10: G, T deprotonated  no H-bonds
pH < 2.3: A, C protonated  no H-bonds
59
P.508
Heat denaturation (热变性)
 Melting (熔解): heat-induced
denaturation
 Melting point (Tm): The
temperature at the midpoint of the
transition (熔点为热变性使DNA的双
螺旋结构失去一半时的温度)
 Tm of DNA: 82~95oC
 Annealing (退火): The process
that a heat-denatured DNA
renatures when slowly cooled
down. (热变性的DNA在缓慢冷却时
可以复性，此过程叫退火)
热变性曲线
熔点
60
P.509
Factors affecting DNA’s Tm
(影响Tm的因素)
 Homogeneity (均一性) of the sample: Homogeneous DNA melts
within a narrow range (均质DNA在较窄的温度范围内熔解).
 Ionic strength (离子强度) of the medium: DNA melts at a lower
Tm and within a broader Tm range when placed at a low ionic
strength (在离子强度较低的介质中，DNA的Tm较低，Tm范围较宽).
 GC content of the DNA:
proportionally related to Tm
(GC含量与Tm成正比)
xG+C = (Tm - 69.3) x 2.44
61
Results of DNA Denaturation
 Dissociation of two paired strands due to the disruption
III. Properties
of the H bonds (由于氢键破裂使两条配对单链分离)
 Decrease in viscosity (粘度下降)
 Increase in A260 (吸光度上升)
 Denaturation (变性)  A260 – Hyperchromic effect
(增色效应)
 Renaturation (复性)  A260 – Hypochromic effect
(减色效应)
 A260 can be used to follow DNA denaturation.
62
P.510
Nucleic Acid Hybridization
(核酸的杂交)


Hybridization: DNA-DNA, RNADNA
Extensively employed in molecular
biology and molecular genetics (广泛
应用于分子生物学和分子遗传学)
 To reveal evolutionary relationships
 To identify a specific gene selectively
 To assay gene expression

DNA fingerprinting
Southern blotting -- DNA
Northern blotting -- RNA
Western blotting -- Protein
63
DNA Fingerprinting/Southern Blotting
限制性核酸内切酶
Lane 1. DNA markers
Lane 2. DNA markers
Lane 3. Suspect 1
Lane 4. Victim
Lane 5. Evidence
Lane 6. Suspect 2
Lane 7. DNA markers
Lane 8. DNA markers
放射性标记
DNA探针
放射自显影64
DNA Polymerase Chain Reaction
(PCR)
P.519
1. PCR can be used to amplify a specific
DNA segment (PCR用于体外扩增DNA).
2. Requirements:
 A target DNA (模板DNA)
 2 synthetic oligonucleotide primers (合成的寡
核苷酸引物)
 Thermostable DNA polymerase (热稳定的
DNA聚合酶)
 4 deoxynucleoside triphosphates (四种dNTP)
3. 3 steps (三个步骤):
1) Heating (加热使DNA变性)
2) Cooling (退火使引物与模板结合)
3) Replication (复制)
65
5. Nonenzymatic transformations
(非酶催化的转化)
III. Properties
1)
2)
3)
4)
5)
Deamination (脱氨基)
Depurination (脱嘌呤碱)
Alkylation (烷基化)
Radiation (辐射)
Reactive chemicals of industrial activity
(化学有害物质)
6) Oxidative damage (氧化作用)
66