Nucleic acid chemistry part 1 : Some aspects of chemical and enzymatical modification of nucleic acids

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Nucleic acid chemistrypart 1 Some aspects of
chemical and enzymatical modification of
nucleic acids
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Chelating agents
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NTA nitrilotriacetic acid (chelating Ca2, Fe2,
Cu2, ...) In contrast to EDTA, it is easily
biodegradable. Carcinogenic in different animal
model systems, hence possibly carcinogenic to
humans. A modified NTA is used to immobilize
nickel to a solid support, and used in the
His-tag method to purify recombinant proteins,
with 6-His at the C-terminus.
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Intercalation
Ethidium bromide
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UV absorption curve of double-stranded DNA
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Spectra of the four individual nucleotides A,
C, G, T
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1 A260 of DNA 50 mg/ml 1 A260 of RNA 40
mg/ml Extinction coefficient, single-stranded
DNA e260 6100 mol-1 x L-1 relative
contributions A gt G gt T gt
C 1.52 1.21 0.84
0.705 The shape of the absorbance curve of
Adenine resembles best the one of large DNA (is
somewhat thinner) the curves of Guanine,
Thymine and Cytosine are more irregular, making
an overall DNA curve a bit broader, but remaining
nicely symmetrically shaped with a maximum close
to 260 nm. There is a dip between 220 and 230
nm. Above 320 nm there is no more absorbance,
and below 220nm (in the far UV) the solution
becomes opaque. At 260 nm, the UV absorption
(OD, optical density) of single stranded DNA is
30 to 40 higher than when base-paired into a
double helix.
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At 280, DNA absorbs about 50 less than at 260
nm, hence DNA has an 260/280 absorbance ratio of
2. (Above 1.7 a sample is considered being
pure.) Proteins have a maximum at 280 nm
(especially due to the aromatic side- chains).
Contamination with protein will lower the
260/280 ratio below 2, although this usually
requires a high level of contaminating protein.
In contract, contamination of protein samples
with (small amounts of) DNA (or RNA) are readily
detected. Other UV-absorbing substances, e.g.
phenol may disturb concentration measurements
substantially. With shorter strands, the A260
value lowers Nucleic Acids A260
(e.c.L) Double-stranded DNA 50 Single-stranded
DNA or RNA (gt100 nucleotides) 40 Single-stranded
oligos (60100 nucleotides) 33 Single-stranded
oligos (lt40 nucleotides) 25
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Protein contamination and the 260280 ratio The
ratio of absorptions at 260 nm versus 280 nm is
commonly used to assess DNA contamination of
protein solutions, since proteins (in particular,
the aromatic amino acids) absorb light at 280 nm.
The reverse, however, is not true it takes a
relatively large amount of protein contamination
to significantly affect the 260280 ratio in a
nucleic acid solution. 260280 ratio has high
sensitivity for nucleic acid contamination in
protein protein nucleic acid 260280
ratio 100 0 0 0.57 95 5 1.06 90 10
1.32 70 30 1.73 260280 ratio lacks
sensitivity for protein contamination in nucleic
acids (table shown for RNA, 100 DNA is
approximately 1.8) nucleic acid protein
260280 ratio 100 0 2.00 95 5 1.99
90 10 1.98 70 30 1.94 This difference
is due to the much higher extinction coefficient
nucleic acids have at 260 nm and 280 nm, compared
to that of proteins. Because of this, even for
relatively high concentrations of protein, the
protein contributes relatively little to the 260
and 280 absorbance. While the protein
contamination cannot be reliably assessed with a
260280 ratio, this also means that it
contributes little error to DNA quantity
estimation.
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Other common contaminants Contamination by
phenol, which is commonly used in nucleic acid
purification, can significantly throw off
quantification estimates. Phenol absorbs with a
peak at 270 nm and a A260/280 of 1.2. Nucleic
acid preparations uncontaminated by phenol should
have a A260/280 of around 2. Contamination by
phenol can significantly contribute to
overestimation of DNA concentration.
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5-bromo uracil 5-methyl cytosine 5-hydroxymethyl
cytosine 5-hydroxymethyl thymine 5-hydroxymethyl
thymine, glucosylated (1 or 2) etc.
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Western alphabet A B C D E F G H I J K L
M N O P Q R S T U V W X Y Z
The names "nucleoside" for pentose base,
and "nucleotide" for phosphate pentose
base were introduced by P.A.Levene W.A.Jacobs
in 1909.
Nomenclature of nucleosides nucleotides
(nucleoside phosphates) (deoxy)adenosine
(deoxy)adenosine phosphate or
(deoxy)adenylate (deoxy)guanosine
(deoxy)guanosine phosphate or (deoxy)guanylate (d
eoxy)cytidine (deoxy)cytidine
phosphate or (deoxy)cytidylate (deoxy)thymidine
(deoxy)thymidine phosphate or
(deoxy)thymidylate (deoxy)uridine
(deoxy)uridine phosphate or (deoxy)uridylate One-l
etter symbols A, G, C, T (originally U for
uridine in RNA, now also T accepted as symbol)
(or dA, dG, dC, dT) for RNA or DNA with inosine
as nucleoside (hypoxanthine as base) symbol
I. The symbol for any nucleoside is N (X for
an undefined (or unknown) nucleoside)
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for other positions with multiple choices
following standard symbols R A or G
(puRine nucleoside) Y T or C (pYrimidine
nucleoside) S G or C ('Strong' basepair) W
A or T ('Weak' basepair) M A or C (aMino
functional group) K T or G (Keto functional
group) B C or T or G ( no A) D A or T
or G ( no C) H A or C or T ( no G) V
A or C or G ( no T)
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3'-deoxyribonucleoside triphosphate
cordicepin triphosphate is another analog.
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base pairing
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At the isotopic level   Alternative isotopes
(underlinedstable isotope) or radioactive
labeling (b-radiation) 31P ltgt 32P,
33P 12C ltgt 13C, 14C 14N ltgt 15N H ltgt T
(3H) 32S ltgt 35S 127I ltgt 36 other isotopes
(127I is the only stable iodine
isotope) 135I has a half-live less than 7
hrs, too short for use in biology
129I has a half-live of 15.7 million years all
others half-lives less than 60 days) ltgt
125I, 131I. (Also 123I and 124I can be used but
are not b- emitters also shorter
half-lives) Radioactive isotopes often used in
molecular biology b--radiation half-life ma
x energy average energy 32P 14.3 days 1.710
MeV 0.70 MeV 33P 25 days 0.249 MeV 0.0769
MeV 35S 87.4 days 0.1673 MeV 0.0492
MeV 3H 12.3 years 0.156 MeV 0.050
MeV 14C 5,730 years 0.018 MeV 0.0055
MeV Other decay 125I 59.4 days (electron
capture) 131I gt still occasionally used
half-life 8 days (b- g emitter)
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Chemical hydrolysis DNA - is relatively stable
in alkali (some cleavage of A and C rings occurs
upon prolonged incubation in 1M
NaOH at 90C) - is acid labile loss of the
base (cleavage of the glycosidic bond) - DNA gt
labile than RNA - purines are lost more easily
than pyrimidines - in formic acid
diphenylamine depurination reaction (named
Burton degradation) gt leading to
oligopyrimidines (also named apurinic acid) -
methylation at G (and much less at A) with
dimethylsulfate, followed by treatment with
piperidine also leads to strand cleavage at
purine nucleotides - in hydrazine
(hydrazinolysis) formation of oligopurines RNA
- is hydrolyzed in alkali to mononucleotides,
via a 2-3 cyclic phosphate intermediate - most
typically, the 2-3 ends are sensitive to
cleavage by periodate (which targets
cis-diols) and may subsequently undergo a
b-elimination (leading to a 3 phosphate end)
or be derivatized by an alkylamine (and sodium
borohydride NaBH4).
(primary structure)
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Formation of apurinic sites in DNA. In the
presence of acid, the N-glycosyl bond of
deoxyadenosine (or deoxyguanosine) is
hydrolyzed, leading to the formation of
the apurinic site and adenine (or guanine).

Reaction of thymidine with hydrazine
(similarly with cytidine)
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acid pH 3-4 loss of purine from the
glycosidic bonds gt then with an
amine cleavage of the apurinic
sites (originally diphenylamine was used by
Burton in the so-called Burton degradation) (othe
r amines may be used, e.g. piperidine (Maxam
Gilbert), or the use of NaOH) at more highly
acidic pH total hydrolysis of DNA in
principle, DNA is stable in alkali (though
obviously denatures to single strands !) but
heating in 1M NaOH leads to partial (random)
fragmentation at A's (and to an even lesser
extent at C's (10 relative to A))
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Formation of apurinic sites in DNA after
alkylation of deoxyadenosine and of
deoxyguanosine by dimethylsulfate.
strand cleavage by heating in 1M piperidine
see Chemical Degradation Sequencing
according to A.Maxam W.Gilbert
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Alkaline hydrolysis of RNA
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Oxidation of the 3 terminal nucleoside of RNA
(cis diol) with sodium periodate and subsequent
derivatization with an alkylamine. (R
CH3(CH2)n- or NH2(CH2)n-)
or b-elimination, leaving a 3'-phosphate
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The amino groups of the bases are targets for
deamination or modification to Schiff bases
Deamination of adenine-, cytosine- and
guanine-containing nucleosides (r ribose or
deoxyribose)
Reaction of aldehydes with cytidine, adenosine
and guanosine to form Schiff base adducts.
(r ribose or deoxyribose R H
or CH3(CH2)n- )
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De-amination at NH2 positions - with
NaNO2 at around pH 4.5 (via diazonium
intermediate derivatives). - rate
ratios of 126 for GAC A gt I
(mutagenic) base-pairing to C at replication C
gt U ( ) base-pairing to
A G gt X (inactivating) I and U
mimic G and T, respectively.   Reaction with
hydroxylamine (NH2OH) - target position
C4 in cytosine results in hydroxylation of the
amino group (and the CN double bond) under
mild conditions (leads to pairing to A)
- results in deamination to U under more
vigorous conditions. - in vitro
treatment of DNA gt mutagenesis. In both cases
leading to CG gt TA   Reaction with Na-bisulfite
(NaHSO3) - deamination of C (to U) is
thousandfold faster than of purines. gt same
transition point mutations as obtained with
hydroxylamine. - is used in vivo and in
vitro. - with R-NH2 and R(NH2)2 gt
4-alkyl-cytosine and 4-(aminoalkyl)-cytosine gt
possibility of attachment of reporter molecule
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Sodium bisulfite-catalyzed deamination of
cytidine to uridine in aqueous solution, and
transamination of cytidine to 4-alkylcytidine or
4-amino-alkylcytidine (r ribose or deoxyribose
R CH3(CH2)n- or NH2(CH2)n-).
(not with 5-methyl-C or 5-hydroxymethyl C)
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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
the preferential target O6-alkylation
- also alkylation of the phosphate moiety
(acidic target!) gt phosphotriester gt
subsequently in strong alkali either loss of
alkyl group or strand cleavage (in strong
acidic conditions regeneration of the
phosphodiester)   Two kinds of reagents
- monofunctional e.g. ethylmethanesulfonate
gt puts a methyl group on G (at N7 and
O6) gt causing faulty pairing with T
- bifunctional e.g. nitrosoguanidine,
mitomycin, nitrogen mustard gt cross-links
the DNA strand faulty region excised by
DNase gt both point mutations and deletions
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nucleophilic centres
7
6
7
1
3
3
4
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Alkylation of deoxyguanosine at basic pH to form
O6-methyldeoxyguanosine.
Alkylation of the phosphodiester internucleotide
bond by ethylnitrosourea.
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Reaction of guanosine with glyoxal (r ribose
or deoxyribose)
Reactions with aldehydes, dialdehydes
- aldehyde formaldehyde form Schiffs bases
(or alike) (see also above) gt derivatives at
exocyclic NH2 groups (C, A, G) gt changes
hydrogen bonding abilities of the bases gt also
cross-linking between bases in ds-DNA  
- dialdehyde glyoxal, kethoxal selective
reaction at G gt formation of an adduct, unable
to base-pair (between C and G)
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Introduction of modifications by filling-in or
copying reactions with DNA or RNA polymerases
and modified dNTPs or rNTPs (or ddNTP to add a
single nucleotide) to introduce modified
nucleotides.
See also in other parts
Filling-in of a recessed 3 OH by polymerase.
Nucleophilic attack onto the a-phosphate. Releas
e of pyrophosphate. Remember antiparallel
orientation of the complementary strands !
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Enzymatical tagging with biotin or digoxigenin
by polymerase reactions. (see also in other
parts)
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Requirements characteristics of the
polymerase exonucleolytic activities ?
(5'gt3' , 3'gt5') processivity ? (cfr. later
"exonucleases") substrates? standard
dNTP ? rNTP ? (Mn2 dependent?) ?
thiophosphate variants ? ? biotinylated
variants ? ? digoxigenin-modified variants ?
? fluorescent modifications ? ? etc...
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What is processivity ? the ability of an
enzyme to repetitively continue its
catalytic function without dissociating from its
substrate. with DNA polymerases the average
number of nucleotides added by a DNA polymerase
enzyme per association/dissociation with the
template. DNA polymerases involved in DNA
replication tend to be highly processive, while
those involved in DNA repair tend to have low
processivity. In vitro, with low processivity
enzymes, more uniform elongation of the DNA
mixture in the sample will be obtained. With
high processivity, there will be more
versatility in chain lengths.
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Alkaline phosphatases dephosphorylation
(phosphomonoesterases) - remove
phosphate from 5-, 3- and 2-ends of nucleic
acid and other phosphomonoester compounds
(including proteins, BCIP, etc.) -
Zn(II) metalloenzymes, which hydrolyze the
monoester via a phosphorylated serine-intermediat
e - BAP, CIAP, SAP stability
versus lability BAP extremely stable,
difficult to inactivate E. coli
enzyme (removed by SDS-phenol after treatment
at gt60C) CIAP calf intestinal enzyme, easier
to inactivate (although still extraction
needed to remove it completely) SAP from
Pandalis borealis (an arctic organism)
completely inactivated by heating at 65C
- inhibition by inorganic phosphate and
chelators of metal ions (EDTA, EGTA)
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Some other specific enzymatic modifications
(not exam material)
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DNA ligases reaction mechanism
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or
DNA ligation mechanism R ribose A adenine N
nicotinamide NMN nicotinamide
mononucleotide E enzyme (ligase)
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Pyrophosphatase e.g. tobacco acid
pyrophosphatase (TAP) gt cleaves phosphoanhydric
bonds e.g. in dNTP, rNTP, CAP structures,
CAP-structure of eukaryotic mRNA
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T4 polynucleotide kinase 5'-phosphorylation
- transfers the g-phosphate of ATP onto
the 5 end (onto a 5-OH, or by an
exchange reaction) - applications
- enables ligations (with e.g. synthetic
fragments) - 5-terminal labelling /
tagging
Terminal deoxynucleotidyl transferase (TdT)
- polymerisation without copying
- monomeric enzyme that adds
5'-mononucleotides to a 3'-OH end of DNA
- may be ss or ds minimal starter
(p)N-N-NOH - substrates dNTP,
rNTP, ddNTP, - a single (d)NTP substrate will
yield a (3-)homopolymic tail - ddNTP as
substrate makes a single addition, also
cordicepin triphosphate ( 3'-deoxy-adenosinetr
ifosfaat) or rNTP followed by alkali (!! in
which case there is an extra phosphate !!)
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Exonucleases
What is processivity ? the ability of an
enzyme to repetitively continue its
catalytic function without dissociating from its
substrate. (see above, DNA polymerases ) With
exonucleases low or no processivity gt more
uniform stepwise degradation of the substrate
high processivity gt more heterogenous
mixture of sizes
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- exonuclease III (of E.coli) - active in the
excision-repair pathway - 31 KDa
monomer globular protein (gene xth) requires
Mg2 - substrate is only ds DNA
active on 5 extensions of ds DNA active
on blunt ends of ds DNA also extending
nicks to gaps not active on single-stranded
DNA but may be active on 3 extensions not
longer than 3 nucleotides or 4
nucleotides if the terminal one is a C
- removes stepwise pN from the 3-OH ends
(or 3'-phosphate ends , since it has some
intrinsic 3-phosphatase activity) - displays
some sequence dependence C gt A T gt G
- end-product two half-strands
(until they fall apart) - not
processive or processive, depending on the
reaction conditions - very efficiently
controllable in time or by temperature gt
progressive deletions (under conditions of low
processivity) - resistant against
thiophosphate linkages (e.g. fill-in one end with
a a-S-dNTP) - other activities
AP-endonuclease (at apurinic and apyrimidinic
sites) RNaseH
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- exonuclease VII (of E.coli) - heterodimer
proteïn 2 genes xseA and xseB
- active on ssDNA from both (3' 5') ends
- shortens ss ends of ds fragments but
not useful to make blunt ends (involved in
repair mechanisms and homolous recombination)
- not active onto internal loops
(hence clearly an exonuclease) -
not sensitive to EDTA (no divalent metal ion
required) - processive -
under limiting conditions gt rather long
oligonucleotides (gt 100 nt) are produced
initially, then further hydrolysed to sizes
of 2-12, but no mononucleotides ! - there
may be a phosphate group at either 5'- or
3'-end - applications (in vitro) - removal
of ss regions - rapid destruction of excess
primers following PCR - (earlier study of
exon intron structures)
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- bacteriophage l exonuclease - most active
on ds DNA with a 5' phosphate (50-250 times
better than on ss DNA) - preferred substrate is
blunt-ended, 5'-phosphorylated (but the reaction
mechanism (in initiation) seems to be quite
complex) - removes pN from the 5' end (if
5'-phosphate, greatly reduced rate at 5'-OH
ends) - highly processive - requires Mg2 -
not active on a nick or a gap - phage T7
exonuclease (phage T7 gene 6 product, discovered
10 years after l exonuclease) - removal of
5'-mononucleotides from the 5' end - active on
ds DNA, regardless of 5'-phosphate or 5'-OH.
(Barely active on ss DNA) - also initiates at
nicks and gaps in a duplex DNA - seems also to
degrade DNA and RNA of DNA/RNA hybrids but is
not active on either ds or ss RNA. - low or no
processivity gt more uniform degradation than
with l exonuclease - M2 absolutely required (5
mM Mg2 or 1 mM Mn2) also a sulfhydryl agent
(DTT)
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- phage T5 exonuclease (phage T5 gene D15 gene
productfound 10 years after l exonuclease) -
initiates nucleotide removal from 5' ends, gaps
and nicks, of linear and circular ds DNA. -
degradation in 5' to 3' direction - supercoiled
DNA (cccDNA) is not degraded. - application
degradation of linear ssDNA, dsDNA or
nicked plasmid DNA while preserving
supercoiled plasmid DNA. - ss DNA endonuclease
activity is suppressed by lowering Mg2
concentration to less than 1mM Many other
exonucleases have been described, some of which
have also been further developed (and
commercialized) for DNA manipulations. E.g.
thermoresistant enzymes. Known and used since
the 1960-70's, as they are active on DNA and RNA,
are the following - snake venom
phophodiesterase cleaves off pN
- exonucleolytically from a 3'-OH of DNA or
RNA, ss or ds - calf spleen phosphodiesterase
cleaves off Np Up gt Gp Ap gtgt Cp
- exonucleolytically from a 5'-OH of DNA
er RNA, particularly on ss substrate inhibited
by secondary structure in ss RNA or DNA
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- DNaseI (bovine pancreas) gt nicking
(dsDNA) - nicking is quite random
produces a 5'-phosphate 3'-OH
- Mg2 required for nicking with Mn2
instead of Mg2 gt second cleavage in the
opposing position of the complementary strand
ds cleavage - with cccDNA nicking
is only once (gt ocDNA) in the presence of
ethidium - application 'nick
translation' labelling and tagging -
Nuclease S1 Mung bean nuclease gt cleave
single strands endonucleolytically
- active on both DNA and RNA (if ss)
- optimal activity around pH 4 to 5 yields
5'-phophate 3'-OH - Zn2 required
- 'breathing' of the ends may lead
to some extra shortening of the ds terminal parts
gt more difficult to control with
nuclease S1 than with mung bean nuclease
- excise loop structures (confirming the
endonucleolytical mechanism)
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- ribonucleases (crystallized very pure
endoribonucleases) - base-specific
cleavages by RNase T1 (following Gp), RNase A
(Yp), RNase U2 (Rp), RNase T2 (Np) gt
(oligo)nucleotides with a
3'-terminal phosphate) - hydrolysis
via a 2'-3' cyclic intermediate (opening is the
rate-limiting step) - ring opening
only by cleavage of the 2' linkage gt only 3'-P
oligonucleotides - cleaving
preferentially in single-stranded regions hence
partial digestions do not generate genuinely
random fragments, but more (secondary)
structure-related overlaps. - RNase
T2 some preference for adenylate linkages, but
full cleavage produces 3'-mononucleotides
(Np) - RNase H (hybridase) breaks the RNA
strand in DNARNA heteroduplexes
wide occurrence in nature - enzymes from
bacteria and eukaryotic cells are
endoribonucleases - viral enzymes are (also?)
exonucleases that stepwise remove
5'-mononucleotides from both the 5' and 3'
end - a bond between a ribo- and a
deoxyribonucleotide is not cleaved by the
bacterial RNaseH, but is cleaved by the viral and
eukaryotic enzymes.
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