KEY CONCEPTS
* Scientists long assumed
that any DNA mutation
that does not change
the final protein
encoded by a gene is
effectively "silent".
* Mysterious exceptions
to the rule, in which
silent changes seemed to
be exerting a powerful
effect on proteins, have
revealed that such
mutations can affect
health through a
variety of mechanisms.
* Understanding the
subtler dynamics of how
genes work and evolve
may reveal further
insights into causes and
cures for disease.
úterý, 13. října 2009
Silence in the code
The genetic code, which governs how a cell translates DNA
instructions via RNA into functional proteins, is unusual in that it is
redundant. Genes "written" in RNA nucleotides spell out the sequence of
amino acid in an encoded protein using three-letter words called codons
that correspond to one of 20 amino acids. With an alphabet of four
nucleotide bases, 64 codon triplets are possible ­ resulting in several
codons that specify the same amino acid. A DNA mutation that
changes one of these codons to its synonym should therefore be
"silent" in protein terms.
Translation to protein
In the cellular cytoplasm, ribosomes unfold and read the mRNA
and produce the encoded amino acid chain with the help of transfer
RNA (tRNA) molecules. Each tRNA delivers a single amino acid to the
ribosome, binding to the corresponding mRNA codon to confirm that
the correct amino acid is being added. The growing amino acid chain
begins folding into its three-dimensional protein shape even as it is
still forming.
úterý, 13. října 2009
Synonymous codons may specify the same amino acid, but a mutation that changes
one codon to its synonym can alter a gene's encoded message if it interferes with
the cell's editing of mRNA. Many diseases are caused by such editing errors, and a
gene involved in cystic fibrosis illustrates how even so-called silent mutations can cause
a gene's protein meaning to change.
Normal RNA splicing. The raw RNA transcript of a
gene contains exons, which encode amino acids, and
long noncoding intron segments that must be edited
out of the final mRNA. Within each exon, short
nucleotide sequences act as exonic splicing
enhancers (ESE) that flag the boundaries of the
exon to cellular editing machinery. The binding of
splicing regulatory (SR) proteins to enhancer sites
directs "spliceosome" proteins to both ends of an
intron, which they excise from the transcript,
before joining the exon ends together.
Exon skipping. Single-nucleotide synonymous changes
in an exon can render splicing enhancer sequences
invisible to the splicing machinery, causing an entire
exon to be left out of the final mRNA.
Protein altering. Mutations in the cystic fibrosis transmembrane-conductance receptor (CFTR) gene that
disable the receptor protein are implicated in cystic fibrosis and several other related disorders. In an
experiment to test whether silent mutations could also affect the CFTR protein, scientists induced singlenucleotide
mutations, one by one, to create synonymous codons in CFTR exon 12, then analyzed the resulting
proteins. The six synonymous mutations shown (one quarter of those tested) each caused exon 12 to be
skipped during mRNA editing, yielding a truncated CFTR protein.
úterý, 13. října 2009
úterý, 13. října 2009
MUFFLED MESSAGE
A synonymous mutation was found to affect pain sensitivity by changing the amount of
an important enzyme that cells produced. The difference results from alteration in the
shape of mRNA that can influence how easily ribosomes are able to unpackage and read
the strand. The folded shape is caused by base-pairing of the mRNA´s nucleotides;
therefore, a synonymous mutation can alter the way nucleotides match up.
úterý, 13. října 2009
2009-10 2.ep PŘEDNÁŠKA 30.9.09
Chemie, struktura a interakce nukleových kyselin
Složky nukleových kyselin
úterý, 13. října 2009
!!
Pyrimidinové báze
úterý, 13. října 2009
!!
Purinové báze
úterý, 13. října 2009
úterý, 13. října 2009
Neobvyklé báze a
nukleosidy
vyskytují se např v tRNA;
jaké další v chromosomálních DNA
(i) prokaryotních a
(ii) eukaryotních buněk?
(iii) v DNA virů?
Může se v DNA vyskytovat uracil?
úterý, 13. října 2009
Neobvyklé báze
a nukleosidy v tRNA
Pseudouridin
úterý, 13. října 2009
Neobvyklé báze v DNA bakteriofágů
úterý, 13. října 2009
Deaminace cytosinu
(C - U)
Hydrolytická nitrosativní
Zbytky uracilu v DNA, vznikající
v důsledku nesprávné inkorporace
nebo díky deaminaci cytosinu, jsou v
lidském organismu odstraňovány pomocí
uracil-DNA glykosylásy,
která je jedním z nejúčinnějších
enzymů v systému reparujícím DNA
(base-excission DNA repair)
úterý, 13. října 2009
Uracil v lidské DNA může
představovat mutační zátěž
nebo nástroj pro odlišení nebo
degradaci DNA
úterý, 13. října 2009
Sugar puckering
Číslovaní uhlíků ve zbytcích cukru:
1', 2' ....5' na rozdíl od bází 1, 2, ... 5, 6 ..
úterý, 13. října 2009
Nucleosides/nukleosidy
Guanine riboside
Guanosine
Thymine deoxyriboside
! beta-glykosidická vazba
(obě vazby na stejné straně kruhu cukru)
úterý, 13. října 2009
Nucleotides/nukleotidy
Ionization of AMP
AMP
cyclic p
cUMPCMP
bis- x di-phosphates (e.g. ADP)
5'-TAGGTCGA-3'
3'-ATCCAGCT-5'
Cp (C-3') x pC (C-5')
UpUp U-Up UpU
2 fosfáty na jedné pentose
úterý, 13. října 2009
Ionization of AMP
BUT, pK of base residues
in DNA may significantly differ!
úterý, 13. října 2009
DNA base pairing
úterý, 13. října 2009
úterý, 13. října 2009
base stacking
antiparallel x parallel strands
glycosidic bond: ANTI
HYDRATION
~70 H20/bp
Between pH 5 and 9
DNA is a polyanion
with a single negative
charge per nucleotide
úterý, 13. října 2009
úterý, 13. října 2009
úterý, 13. října 2009
úterý, 13. října 2009
25
If you want to do research into the DNA electrochemistry it is not sufficient to
know well methods of electrochemical analysis, you have to know something
about DNA. Since the beginning of the 1950's DNA has become one of the most
studied objects. It has been studied not only by chemists, physicists and
biologists but also by MD's, anthropologists, biotechnologists, etc. because of its
utmost biological importance and also for its interesting properties such as selfcomplementarity
of its strands, strand separation and restoration of its doublehelical
structure, etc.
I therefore decided to start my lecture series with DNA
Electrochemistry of DNA
úterý, 13. října 2009
DNA electrochemistry
Co je polarografie?
Kdo ji objevil?
Moderní elektrochemické metody
Může být elektrochemie užitečná při
výzkumu nukleových kyselin a bílkovin?
úterý, 13. října 2009
d.c. polarography
1922
Oscillographic polarography at controlled a.c
(cyclic a.c. chronopotentiometry)
complete analyses on a single drop 1941
Nobel Prize 1959
Jaroslav Heyrovský 1890-1967
úterý, 13. října 2009
Electrodes
A number of electrodes have been used in electrochemical NA and protein studies, such
as liquid mercury and solid mercury-containing electrodes (such as film and solid
amalgam, incl. dental amalgam electrodes), carbon, gold, indium-tin oxide, silver, etc. Only
with mercury-containing and carbon electrodes well-behaved electroactivity was
observed. Mercury electrodes and most of the solid electrodes greatly differ in their
potential windows
Hg electrodes thus suits better for reductions while solid electrodes (e.g. carbon, Au,,,)
are better for oxidation processes. Material of the electrode is also very important.
Hydrophobicity/hydrophilicity as well reactive atoms and functional groups may greatly
affect adsorption of DNA and proteins
-2 V Hg 0 V
Carbon,Au,Ag, Pt....
-1V +1 V
úterý, 13. října 2009
DNA and RNA are Electroactive Species
producing faradaic and other signals on interaction with electrodes
Cytosine (C)
Adenine (A) A, C, G are reduced at MERCURY electrodes
Guanine (G) reduction product of guanine is oxidized back to G
All bases (A, C, G, T, U) yield sparingly soluble compounds with
mercury and can be determined at concentration down to 10-11
M.
Solid amalgam electrodes can be used instead of the mercury drop electrodes.
____________
A and G as well as C and T are oxidized at CARBON electrodes
_________________________________________________________
PEPTIDE NUCLEIC ACID (PNA) BEHAVES SIMILARLY TO DNA AND RNA
_________________________________________________________________________________________________
Microliter volumes of the analyte are sufficient for analysis
_____________________________________________________________
Electroactive Labels can be Introduced in DNA
Fojta, M., et al.. (2007): ,,Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes. Anal. Chem.
79, 1022-1029
Trefulka, M., et al. (2007): Covalent labeling nucleosides, RNA and DNA with VIII- and VI-valent osmium complexes. Electroanal. 19,
1281-1287
úterý, 13. října 2009
Progress in genomics affects electroanalysis
LOW DENSITY CHIPS
Many areas of science are influenced by the fast
development of the genomics and by the success of the
Human Genome Project.
Classical sequencing of individual human genomes with
3x109
base pairs is too difficult.
Sequencing by DNA hybridization is gaining importance
Relatively expensive DNA hybridization ARRAYS with
optical detection are currently applied in research labs
It is believed that electrochemistry can complement the
optical detection providing new LESS EXPENSIVE
hybridization detection for decentralized DNA analysis
in many areas of practical life
úterý, 13. října 2009
31
END-LABELING of DNA and RNA
Electroactive labels such as ferrocene, daunomycin, viologen, thionine, etc.
were covalently bound to DNA to obtain electrochemical signals closer to
zero charge and/or to increase the sensitivity of the analysis. These labels
are expensive and can hardly be used for labeling of longer NAs, such as
plasmid DNAs.
Osmium tetroxide complexes with nitrogen ligands (OsVIII
,L) can be used for
DNA labeling regardless of the DNA length, in an average biochemical or
biological laboratory without any special equipment. DNA-OsVIII
,L adducts
produce redox signals at mercury, amalgam, carbon and gold electrodes; in
addition, electrocatalytic signals can be obtained at mercury and amalgam
electrodes. Multiple labels can be easily introduced.
T T T TT
Os OsOsOsOs
specific sequence
With six-valent Os(VI)L ribose residue
can be modified
Trefulka, M., et al. (2007): Covalent labeling of nucleosides, RNA and DNA with
VIII- and VI-valent osmium complexes. Electroanalysis 19 (No.12) 1281-1287.
úterý, 13. října 2009
Electrochemical sensors/detectors for DNA hybridization
Single-Surface Technologies:
COVALENTLY BOUND LABEL
ENZYME, etc.
A Heeger
J K Barton
Surface-attached molecular beacons
In the last decade nucleic acid electrochemistry was oriented predominantly to DNA
sensors for (a) DNA hybridization and (b) DNA damage.
This trend has been accompanied not only by interesting discoveries but also by a
number of poor papers lacking the necessary control experiments,claiming sequence
detection without PCR amplification but using synthetic oligos as target DNA, etc.
úterý, 13. října 2009
Double-surface technique
Few years ago we proposed a new technique in which (in difference to previous techniques) DNA
hybridization is separated from electrochemical detection. Optimum properties of the
hybridization surface (H) and the detection electrode (DE) are not identical. We used magnetic
beads optimized for hybridization as surface H and chose optimum DE for the given electrode
process.
With spherical magnetic beads non-specific binding of NAs is minimized. 20 microL of the bead suspension
gives 3 to 7 cm2
area. Beads can be incorporated into microfluidic systems and chips
With single-surface
techniques analysis of long
DNA target molecules and
in large excess of
noncomplementary DNA
may be difficult
úterý, 13. října 2009
Electrochemical sensors for DNA hybridization
At present both single- and double-surface techniques can be
used for DNA sequencing of longer oligonucleotides and PCR
products.
Electrochemical detection of point mutations is also possible.
Optimization of the procedures are now necessary to develop
commercially successful devices.
Challenges:
1) Sequencing eukaryotic DNA without amplification (by PCR).
Great sensitivity and specificity of the analysis is required
2) Development of electrochemical sensors for DNA-protein
protein-protein interactions for proteomics and biomedicine
úterý, 13. října 2009
Elektrochemie nukleových kyselin
není omezena jen na sensory.
Může se zabývat např.
- strukturními přechody DNA
(a) v roztoku
(b) na elektrodě
- adsorpcí DNA na elektricky nabitých
površích
- interakcemi DNA
(a) s nízkomolekulárními látkami
včetně mutagenních látek
(b) s bílkovinami (včetně enzymů
(c) sjinými makromolekulami
- stanovením DNA v roztocích
- elektrickými vlastnostmi DNA (např.
vodivost) atd.
S jakými DNA v současnosti zpravidla pracujeme:
úterý, 13. října 2009
The results of the DNA electrochemistry studies and
development of the electrochemical DNA hybridization
sensors in the last decade suggest that these sensors can
complement DNA sensors with optical detection
How and when the DNA electrochemistry begun?
úterý, 13. října 2009
Brdicka's Co-solution
(background electrolyte)
0.5 mg of RNA per ml
of Brdicka's Co-solution
0.5 mg of RNA plus
0.5 mg of BSA per ml
of Brdicka's Co-solution
How did it begin?
1958: all bases, DNA and
RNA are electroactive
1957: NO response of RNA
and DNA on oscillopolarograms
1955 :Adenine is polarographically reducible
at strongly acid pH while other NA bases are
inactive. J.N.Davidson and E.Chargraff: The
Nucleic Acids, Vol.1, Academic Press, New York
1955
1960: Native (ds) and denatured
(ss) DNA yield different responses
H. BERG, Biochem. Z.
329 (1957) 274
E. PALECEK, Naturwiss.45 (1958) 186-187 E. PALECEK, Nature 188 (1960) 656-657
úterý, 13. října 2009
After February 1948 life in Czechoslovakia was
increasingly affected by the stalinist ideology and heavily
controlled by the Party and Government.
Many scientists and scholars were fired from Universities
but some of them got employment in the Institutes of the
Czechoslovak Academy of Sciences established in 1952.
This was possible particularly at the Institutes whose
Directors were influential Party members but serious
scientists.
Science in Czechoslovakia after the IInd World War
úterý, 13. října 2009
Chemistry and Biochemistry of Proteins and Nucleic Acids
Institute of Organic Chemistry and Biochemistry/
Director: F. Šorm
Institute of Biophysics, Brno
Director: F. Hercík
Founded in 1955 for radiobiological research it gradually turned into
an institute devoted mainly to DNA
For a long time we received 50 - 100 US $ for materials/chemicals
per year and Department. The orders of materials from the West
had to be planned 1-2 years ahead
Taking part in meetings in western countries was difficult not only
because of currency problems
B. Keil, B. Meloun, O. Mikes, J. Doskocil, D. Grunberger, A. Holy,
I. Rychlík, J. Ríman, J. Sponar, V. Paces, Z. Sormová, S. Zadrazil
For many years Czech scientists were efficiently isolated from the West
In this respect the situation in Brno was much worse than in Prague
PRAHA/PRAGUE
úterý, 13. října 2009
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
úterý, 13. října 2009
úterý, 13. října 2009
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
úterý, 13. října 2009
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
úterý, 13. října 2009
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
úterý, 13. října 2009
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.
úterý, 13. října 2009
+
+
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 and III
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
úterý, 13. října 2009
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)
úterý, 13. října 2009
RENATURATION OF RNA
AS DETECTED BY DPP
Time dependence
úterý, 13. října 2009
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
úterý, 13. října 2009
úterý, 13. října 2009
51
NPP
dcP
DPP
native denatured
1974
DNA unwinding at negatively charged surfaces
úterý, 13. října 2009
52
In native DNA its NPP responses depended
on the initial potential, Ei
úterý, 13. října 2009
DME
HMDE
TIME TIME
Ered
Ei
E
A1
1s
60 s
III
II
a
b
I
SIGNALAPPLIEDRESPONSEOBTAINED
c
3
1
d
3
3
2
1e
f
E
B
A2
ssDNA
dsDNA
úterý, 13. října 2009
54
Effect of pH on DNA unwinding
úterý, 13. října 2009
55
Effect of nucleotide sequence on DNA unwinding
úterý, 13. října 2009
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
úterý, 13. října 2009
57
úterý, 13. října 2009
58
úterý, 13. října 2009
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
úterý, 13. října 2009
60
denatured DNA
native DNA
peak G
úterý, 13. října 2009
Scheme 1
x
Potential region U (around -1.2 V)
(first seconds) (tens of seconds)
B C
Potential region T
A
úterý, 13. října 2009
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
úterý, 13. října 2009
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 Xray
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 .....
úterý, 13. října 2009
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
úterý, 13. října 2009
65
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.
úterý, 13. října 2009