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4. Chemical modification of DNA
At the atomic level
isotopic (underlined=stable) or radioactive labeling
31
P
=>
32
P, 33P
12
C
=>
13
C, 14C
14
N
=>
15
N
H
=>
T (3H)
radioactive isotopes used decay usually by -radiation
-radiation
half-life
max energy
average energy
32
14.3 days
25 days
87.4 days
12.3 years
5,730 years
1.710 MeV
0.249 MeV
0.1673 MeV
0.156 MeV
0.018 MeV
0.70 MeV
0.0769 MeV
0.0492 MeV
0.050 MeV
0.0055 MeV
P
P
35
S
3
H
14
C
33
chemical changes
P=O => P=S
P-O- => P-S(remember tautomerism !!!)
=> effects on exonuclease activity, polymerase activity, T4-kinase, etc...
=> S32 may be replaced by the radioactive S35 (-emitter)
For other chemical modifications : see chapter 2 : chemical DNA synthesis
At the DNA fragment level
- end groups : phosphate or no phosphate (at 5’, at 3’, at 2’ (in RNA))
- sequence: blunt, protrusions 5’ or 3’
- varying the sequence size
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G. Volckaert
Chemical modification of DNA
12/02/2016
2
Some chemical considerations:
Hydrolysis
- DNA is relatively alkali stable (in contrast to RNA, which is hydrolyzed in alkali
to mononucleotides, via a 2’-3’ cyclic phosphate intermediate)
(in DNA, some cleavage of A and C rings occurs upon prolonged incubation at 90°C)
- but is acid labile : loss of base (glycosidic bond)
- DNA > labile than RNA
- purines are lost more easily than pyrimidines
formic acid + diphenylamine : depurination : leads to oligopyrimidines (Burton
degradation) (also named apurinic acid)
in hydrazine (hydrazinolysis) : formation of oligopurines
Application in chemical degradation sequencing:
chemical reagent => damage of base (modification) => cleavage of strand
- dimethylsulphate (DMS) : methylation at G (N-7)
- heating in alkali: A > C (ring opening)
- KMnO4, OsO4 : reaction at T
- Hydrazine : T and C, reaction at T suppressed in 1M NaCl
- formic acid : depurination
partial reactions (average one hit/molecule)
strand cleavage by heating in piperidine/water
De-amination
- with NaNO2 at around pH 4.5 (via diazonium intermediate derivatives).
- rate : ratios of 1:2:6 for G:A:C
A => I
(mutagenic)
base-pairing to C at replication
C => U
(
base-pairing to A
G => X
(inactivating)
“
)
I and U mimic G and T, respectively.
G. Volckaert
Chemical modification of DNA
12/02/2016
3
Reaction with hydroxylamine (NH2OH)
- target position C4 : results in hydroxylation of amino group (and the C=N
double bond) under mild conditions (which pairs to A) ;
results in deamination to U under more vigorous conditions.
- In vitro treatment of DNA => mutagenesis. In both cases leading to CG => TA
Reaction with Na-bisulfite (NaHSO3)
- deamination of C (to U) is thousandfold faster than of purines.
=> same transition point mutations as obtained with hydroxylamine.
- is used in vivo and in vitro.
- with R-NH2 and R(NH2)2
=> 4-alkyl-cytosine and 4-(aminoalkyl)-cytosine
=> possibility of attachment of reporter molecule
Alkylating agents:
e.g. (m)ethylmethanesulfonates
(m)ethyl N-nitrosoureas
- nucleophilic centres in DNA molecules are the N and O atoms in the bases
- most electron-dense (and hence nucleophilic) : N-7 in G (see above DMS methylation)
- other sites: O6 and N-3 in G (less than N-7)
N-7, N-1, N-3 in A
O4 in T
- in (after) alkali : O6 is preferential target : O6-alkylation
- also : alkylation of the phosphate moiety (acidic target!) => phosphotriester
=> subsequently: in strong alkali : either loss of alkyl group or strand cleavage
(in strong acidic conditions: regeneration of the phosphodiester)
Two kinds of reagents:
G. Volckaert
Chemical modification of DNA
12/02/2016
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- monofunctional: e.g. ethylmethanesulfonate :
=> puts a methyl group on G (at N7 and O6)
=> causing faulty pairing with T
- bifunctional : e.g. nitrosoguanidine, mitomycin, nitrogen mustard
=> cross-links the DNA strand : faulty region excised by DNase
=> both point mutations and deletions
Addition reaction
- is possible with the 5,6 double bond of T, U (and C to a lesser extent)
e.g. with H2 + palladium catalyst
- upon radiation with UV light: formation of T-dimers from adjacent residues
=> excision-repair mechanisms (may lead to errors or deletions)
- ionizing radiation : free radical attack, breaking chain (can likewise lead to
errors or deletions)
Reactions with aldehydes, dialdehydes :
- aldehyde : formaldehyde : form Schiff bases
=> derivatives at exocyclic NH2 groups (C, A, G)
=> changes hydrogen bonding abilities of the bases
=> also cross-linking between bases in ds-DNA
- dialdehyde: glyoxal, kethoxal : selective reaction at G
=> formation of adduct, unable to base-pair (between C and G)
Reaction with periodate
- target: cis-diols, as in RNA (at the 3’-terminus)
=> cleavage of 2’-3’ bond; derivatisation possible with alkylamine
G. Volckaert
Chemical modification of DNA
12/02/2016
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Intercalation :
e.g. with ethidium bromide :
=> modifying helical (and hence also superhelical) topology
- intercalation of one ethidium+ moiety unwinds the helix by 26°
- Van der Waals contacts with the base-pairs above and below.
- increased fluorescence compared to the dye in solution
UV (254 nm) : absorbed by the bases and transmitted to the dye
(302 & 366 nm) : absorbed by the dye itself
=> fraction of energy re-emitted at 590 nm
- binding is reversible, but dissociation is very slow (washing of gel in water is OK !)
=> removal at EtdBr : only by chromatography over a cation-exchange resin
or extraction with isopropanol or n-butanol
- causes microinsertions & microdeletions
- other examples: actinomycin D, terpyridine, daunomycin, acridines, etc.
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G. Volckaert
Chemical modification of DNA
12/02/2016