1958: Nucleic acid bases, DNA and RNA are electroactive
50 years of nucleic acid electrochemistry
...part of the guanine ring important for the anodic signal is near to the surface
whereas the the analogous part of cytosine is hidden inside the DNA double helix
participating in the hydrogen bonding.... (showing a cathodic signal in ssDNA
but not in dsDNA)
E. Palecek, Nature 188 (1960) 656-657
E. Palecek, Fifty years of nucleic acid electrochemistry, Electroanalysis 2009, 21, 239-251.
~700 papers in 2008
OSCILLOGRAPHIC POLAROGRAPHY
At controlled alternating current (constant current chronopotentiometry)
dE/dt
E
ssDNAdsDNA
CA
Ganodic
cathodic
sparingly soluble
compounds with Hg
LITERATURE in 1958: Adenine is polarographically reducible at strongly acid pH while
other NA bases as well as DNA are inactive
J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol. 1, Academic Press, New York 1955
Palecek E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186
Palecek E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656
J Heyrovsky S Ochoa A Kornberg
J. Heyrovsky invented
POLAROGRAPHY in 1922.
After 37 years he was awarded
a Nobel Prize
Nobel Prizes 1959
In difference to most of the electrochemists I met in
the 1960`s and 1970`s, J Heyrovsky was interested in
nucleic acids and he greatly stimulated my polarographic
studies of DNA
D.c. polarography vs. oscillopolarography (OP)
Why d.c. polarography was rather
poor in DNA analysis?
(a) no DNA accumulation at the
electrode
(b) DNA adsorption at negatively
charged DME (~-1.4V) compared to
open current potential in OP
J M at the 40th Anniversary of the
Discovery of the DNA Double Helix
In 1960 when I published my NATURE paper
on electrochemistry of DNA I obtained invitations
from 3 emminent US scientists:
J. Marmur - Harvard Univ.
L. Grossman - Brandeis Univ.
J. Fresco - Princeton Univ.
To work in their laboratories as a postdoc
In 1960 new techniques were sought to study DNA
Denaturation and Renaturation. To those working with
DNA Oscillographic Polarography (OP) appeared as a
very attractive tool. Invented by J. Heyrovsky, it was fast
and simple, showing large differences between the signals
of native and denatured DNA. The instrument for OP was
produced only in Czechoslovakia.
I accepted the invitation by Julius Marmur but for more
than two years I was not allowed to leave Czechoslovakia.
In the meantime JM moved from Harvard to Brandeis Univ.
By the end of November 1962 I finally got my exit visa and
with Heyrovsky Letter of Reccommendation in my pocket
I went to the plane just 24 hours before expiration of my
US visa. Before my departure I sent my OP instrument by
air to Boston. It arrived after 9 months completely broken. I
nstead of OP I had to use ultracentrifuges and
microbiological methods.
oscilak
Julius Marmur discovered DNA
Renaturation/Hybridization and
proposed (in JMB) a new method of
DNA isolation which was widely applied.
His paper was quoted > 9000x.
At the end of my stay at Brandeis I did some
OP experiments which I finished in Brno
and published in J. Mol. Biol. in 1965 and 1966.
+
+
Native DNA
melting melting
quick cooling quick cooling
melted DNA
slow cooling
renaturation
denatured DNA RENATURED DNA
Temperature
premelting
C
D
A, B C D
A260
A B
no CI-2
peak II
CI-2peak II
INCREASING
INCREASING
CI-2
peak II andIII
CI-2
only peak III
no CI-2
peak II
no CI-2
peak II
CI-2
only peak III
CI-2
E
III
II
DNA Premelting and Polymorphy of the DNA Double Helix
B. sublilis and B. brevis DNAs have the same
G+C content and different nucleotide sequence
B. subtilis
B. brevis
J. Mol. Biol.
20 (1966) 263-281
Before my departure to the US I observed
Changes in the polarographic behavior of
DNA far below the denaturation
temperature. These changes were
later called DNA Premelting
POLAROGRAPHIC BEHAVIOR OF dsDNA
At roomand premeltig temperaturse depended on
DNA nucleotide SEQUENCE
1976
What the people said:
Before 1980
No doubt that this electrochemistry
must produce artifacts because we know
well that the DNA double helix has
a unique structure INDEPENDENT
of the nucleotide SEQUENCE
After 1980
Is not it strange that such an obscure technique can
recognize POLYMORPHY
OF THE DNA DOUBLE HELIX?
What the people said
Meeting F. Crick in Copenhagen
and Arhus, 1977 (B. Clark)
RENATURATION OF RNA
AS DETECTED BY DPP
Time dependence
We developed methods of chemical
probing of the DNA structure based
on osmium tetroxide complexes
(Os,L). Some of the Os,L complexes
react with single-stranded DNA but
not with the double-stranded B-DNA.
These methods yielded information about the
distorted and single-stranded regions in the DNA
double helix at single-nucleotide resolution. DNA
probed both in vitro and directly in cells.
Probing of DNA structure with osmium tetroxide complexes
In the beginning of the 1980`s Os,L complexes
were the first electroactive labels covalently
bound to DNA. These complexes produced
catalytic signals at Hg electrodes allowing
determination of DNA at subnanomolar
concentrations
Firsts in Electrochemistry of Nucleic Acids during the initial three decades
1958 DNA and RNA and all free bases are electrotractive
1960-61 assignment of DNA electrochemical signals to bases, relation between the DNA structure and
electrochemical responses
1961 adsorption (ac impedance) studies of DNA (IR Miller, Rehovot)
1962-66 DNA premelting, denaturation, renaturation/hybridization detected electrochemically,
traces of single stranded DNA determined in native dsDNA. Nucleotide sequence affects dsDNA responses
1965 Association of bases at the electrode surface (V. Vetterl)
1966 application of pulse polarography to DNA studies
1967 detection of DNA damage
1967-68 Weak interactions of low m.w. compounds with DNA (P.J. Hilsson, M.J. Simons, Harrow, UK
and H. Berg, Jena)
1974 DNA is unwound at the electrode surface under certain conditions (EP and H.W. Nürnberg, Jülich,
independently)
1976 Evidence for polymorphy of the DNA double-helical structure
For two decades only mercury electrodes were used in NA electrochemistry
1978 Solid (carbon) electrodes introduced in nucleic acid research (V. Brabec and G. Dryhurst, Norman)
1980 Determination of bases at nanomolar concentrations by cathodic stripping
1981-83 Electroactive markers covalently bound to DNA
1986-88 DNA-modified electrodes
Results obtained at: IBP, Brno or elsewhere (author's name is given); the results which have been utilized in the DNA
sensor development are in blue
13
NPP
dcP
DPP
native denatured
1974
DNA unwinding at negatively charged surfaces
14
In native DNA its NPP responses depended
on the initial potential, Ei
DME
HMDE
TIME TIME
Ered
Ei
E
A1
1s
60 s
II
II
a
b
I
SIGNALAPPLIEDRESPONSEOBTAINED
c
3
1
d
3
3
2
1e
f
E
B
A2
ssDNA
dsDNA
16
Effect of pH on DNA unwinding
17
Effect of nucleotide sequence on DNA unwinding
In 1986 we proposed Adsorptive Transfer Stripping Voltammetry (AdTSV) based on easy preparation of
DNA-modified electrodes
AdTSV has many advatages over conventional voltammetry of NAs:
1) Volumes of the analyte can be reduced to few microliters
2) NAs can be immobilized at the electrode surface from media not suitable for the voltammetric analysis
3) Low m.w. compounds (interfering with conventional electrochemical analysis of NAs) can be washed away
4) Interactions of NAs immobilized at the surface with proteins and other substances in solution and influence of
the surface charge on NA properties and interactions can be studied, etc.
ADSORPTIVE STRIPPING
NA is in the electrolytic cell and accumulates at the
electrode surface during waiting
ADSORPTIVE TRANSFER STRIPPING
NA is attached to the electrode
from a small drop of solution (3-10
l)
NA is at the electrode but the
electrolytic cell contains only blank
electrolyte
19
20
21
denatured DNA
native DNA
peak G
Scheme 1
x
Potential region U (around -1.2 V)
(first seconds) (tens of seconds)
B C
Potential region T
A
Figure 19
DNA unwinding at negatively charged Au surfaces was recently
observed by R. Georgiadis et al. and applied in DNA sensors
Heaton RJ, Peterson AW, Georgiadis RM, PNAS 98 (2001) 3701
IFFY stories
On this day 50 years ago, Watson and Crick published their double-helix theory. But, what if...
By Steve Mirsky (2003)
"I am now astonished that I began work on the triple helix structure, rather than on the double
helix," wrote Linus Pauling in the April 26, 1974 issue of Nature.
In February 1953, Pauling proposed a triple helix structure for DNA in the Proceedings of the
National Academy of Sciences (PNAS). He had been working with only a few blurry X-ray
crystallographic images from the 1930s and one from 1947.
If history´s helix had turned slightly differently, however, perhaps the following timeline might be
more than mere musing...
August 15, 1952: Linus Pauling (finally allowed to travel to England by a US State Department that
thinks the words "chemist" and "communist" are too close for comfort) visits King´s College
London and sees Rosalind Franklin´s X-ray crystallographs. He immediately rules out a triple helical
structure for DNA and concentrates on determining the nature of what is undoubtedly a double
helix.
February 1953: Pauling and Corey describes the DNA double helix structure in PNAS .....
24 Glading, R. E., Paper Trade J., 111 (No. 23), 32 (1940).
A PROPOSED STRUCTURE FOR THE NUCLEIC ACIDS
BY LINUS PAULING AND ROBERT B. COREY
GATES AND CkELLIN LABORATORIES OF CHEMISTRY,* CALIFORNIA INSTITUTE OF
TECHNOLOGY
Communicated December 31, 1952
The nucleic acids, as constituents of living organisms, are comparable in
importance to the proteins. There is evidence that they are involved in
the processes of cell division and growth, that they participate in the transmission
of hereditary characters, and that they are important constituents
of viruses. An understanding of the molecular structure of the nucleic
acids should be of value in the effort to understand the fundamental phenomena
of life.
We have now formulated a promising structure for the nucleic acids, by
making use of the general principles of molecular structure and the available
information about the nucleic acids themselves. The structure is not
a vague one, but is precisely predicted; atomic coordinates for the principal
atoms are given in table 1. This is the first precisely described structure for
the nucleic acids that has been suggested by any investigator. The structure
accounts for some of the features of the x-ray photographs; but detailed
intensity calculations have not yet been made, and the structure cannot
be considered to have been proved to be correct.
The Formulation of the Structure.-Only recently has reasonably complete
information been gathered about the chemical nature of the nucleic acids.
The nucleic acids are giant molecules, composed of complex units. Each
unit consists of a phosphate ion, HPO4--, a sugar (ribose in the ribonucleic
CHEMISTRY: PA ULING AND COREY
I de Stevens, G., and Nord, F. F., J. Am. Chem. Soc., 75, in press (1953).
10 Brauns, F. E., Ibid., 61, 2120 (1939).
11 (a) Schubert, W. J., and Nord, F. F., Ibid., 72, 977 (1950); (b) Kudzin, S. F., and
Nord, F. F., Ibid., 73, 4619 (1951).
12 Nord, F. F., and de Stevens, G., Naturwiss., 39, 479 (1952).
'3 Maule, C., Beitrdge wiss. Bot., 4, 166 (1900).
14 Kudzin, S. F., and Nord, F. F., J. Am. Chem. Soc., 73, 690 (1951).
51 Klason, P., Svensk Kem. Tidsk., 9, 135 (1897).
16 Freudenberg, K., Sitzungsber. Heidelberger Akademie Wissensch. (1949), No. 5.
17 Hagglund, E., Chemistry of Wood, p. 344, Academic Press, New York (1951).
18 Vitucci, J. C., and Nord, F. F., Arch. Biochem., 14, 243 (1947).
19 Nord, F. F., and Vitucci, J. C., Advances in Enzymol., 8, 253 (1948).
20 Vitucci, J. C., and Nord, F. F., Arch. Biochem., 15, 465 (1947).
21 Birkinshaw, J. H., and Findlay, W. P. K., Biochem. J., 34, 82 (1940).
22 Byerrum, R. U., and Flokstra, J. H., Federation Proceedings, 11, 193 (1952).
23 Nord, F. F., and Schubert, W. J., Holzforschung, 5, 8 (1951).
24 Glading, R. E., Paper Trade J., 111 (No. 23), 32 (1940).
A PROPOSED STRUCTURE FOR THE NUCLEIC ACIDS
BY LINUS PAULING AND ROBERT B. COREY
GATES AND CkELLIN LABORATORIES OF CHEMISTRY,* CALIFORNIA INSTITUTE OF
TECHNOLOGY
Communicated December 31, 1952
The nucleic acids, as constituents of living organisms, are comparable in
importance to the proteins. There is evidence that they are involved in
the processes of cell division and growth, that they participate in the transmission
of hereditary characters, and that they are important constituents
of viruses. An understanding of the molecular structure of the nucleic
acids should be of value in the effort to understand the fundamental phenomena
of life.
We have now formulated a promising structure for the nucleic acids, by
making use of the general principles of molecular structure and the available
information about the nucleic acids themselves. The structure is not
a vague one, but is precisely predicted; atomic coordinates for the principal
atoms are given in table 1. This is the first precisely described structure for
the nucleic acids that has been suggested by any investigator. The structure
accounts for some of the features of the x-ray photographs; but detailed
intensity calculations have not yet been made, and the structure cannot
be considered to have been proved to be correct.
The Formulation of the Structure.-Only recently has reasonably complete
information been gathered about the chemical nature of the nucleic acids.
The nucleic acids are giant molecules, composed of complex units. Each
unit consists of a phosphate ion, HPO4--, a sugar (ribose in the ribonucleic
84 PROC. N. A. S.
CHEMISTRY: PA ULING AND COREY
which are involved in ester linkages. This distortion of the phosphate
group from the regular tetrahedral configuration is not supported by direct
experimental evidence; unfortunately no precise structure determinations
have been made of any phosphate di-esters. The distortion, which corresponds
to a larger amount of double bond character for the inner oxygen
atoms than for the oxygen atoms involved in the ester linkages, is a reasonFIGURE
6
Plan of the nucleic acid structure, showing several nucleotide residues.
able one, and the assumed distances are those indicated by the observed
values for somewhat similar substances, especially the ring compound
S309, in which each sulfur atom is surrounded by a tetrahedron of four
oxygen atoms, two of which are shared with adjacent tetrahedra, and two
unshared. The O-O distances within the phosphate tetrahedron are 2.32
A (between the two inner oxygen atoms), 2.46 A, 2.55 A, and 2.60 A. The
PROC. N. A. S.92
Triple helix
with bases on the outside and
sugar-phosphate backbone in the
interior of the molecule
My IFFY story:
If L. PAULING had in his lab an oscillopolarograph in
1952 he would never proposed this structure.
Polarography clearly showed that bases must be
hidden in the interior of native DNA molecule and
become accessible when DNA is denatured
26
SUMMARY
Electroactivity of nucleic acids was discovered about 50 years ago
Reduction of bases at Hg electrodes is particularly sensitive to
changes in DNA structure. The course of DNA and RNA denaturation
and renaturation can be easily traced by electrochemical methods
At present electrochemistry of nucleic acids is a booming field,
particularly because it is expected that sensors for DNA
hybridization and for DNA damage will become important tools in
biomedicine and other regions of practical life in the 21st century
DNA-modified electrodes can be easily prepared;
microL volumes of DNA are sufficient of its analysis but
miniaturization of electrodes decreases these volumes to nL.
Sensitivity of the analysis has greatly increased in recent years.
2008-09 3.EP/6. PŘEDNÁŠKA 22.10.08
Chemie, struktura a interakce nukleových kyselin
Fyzikální vlastnosti a izolace DNA
Denaturace, renaturace a hybridizace DNA
Biosyntetické polynukleotidy
V posledních letech jsou k dispozici komerčně dostupné
kolonky využívající imobilizaci DNA na pevném podkladu.
K separaci DNA jsou rovněž používány magnetické kuličky
(magnetic beads)
konec 7.10.09
Characterize your DNA sample:
ds x ss, circular x linear
circular: nicked, oc; covalently closed, cc, cd
linear: cohesive or blunt ends
number of base pairs,
purity: protein, RNA .... content
analytical methods
Denaturation x degradation
aggregation
renaturation/hybridization
+
+
Native DNA
melting melting
quick cooling quick cooling
melted DNA
slow cooling
renaturation
denatured DNA RENATURED DNA
Temperature
premelting
C
D
A, B C D
A260
A B
DNA DENATURATION and RENATURATION/HYBRIDIZATION
J. Marmur and P. Doty
DNA renaturation/reassociation depends on the concentration of the DNA molecules and the time
allowed for reassociation. Often imperfect matches may be formed which must again dissociate to
allow the strands to align correctly. C0t value of DNA is defined as the initial concentration C0 in moles
nucleotides per Litre multiplied by time t in seconds. C0t reflects complexity of DNA. Methods: S1,
hydroxyapatite - dsDNA binds more strongly
Microbiologist, biochemist and molecular biologist
Julius Marmur ­ dicovered renaturation of DNA
22 March, 1926 Bialystok (Poland) ­ 20 May, 1996 New York, NY
Oswald Avery 1944 - DNA is a genetic material
(Rockefeller Institute, New York, NY)
Rollin D. Hotchkiss
Julius Marmur
1993
Syntetické oligonukleotidy
Dr. L. Havran, 1. předn.
Důležité modely vlivu
sekvence nukleotidů
na vlastností DNA
nukleosid-difosfáty
nevyžaduje primer ani matrici
nukleosid-trifosfáty
poly(A)
poly(rC)
poly(dG)
poly(U)
poly(rT)