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Infrared spectra of DNA: Correlation of theoretical and observed frequencies
JOURNAL
OF
MOLECULAR
Infrared
SPECTROSCOPY
41, 195-202 (1972)
Spectra of DNA: Correlation of Theoretical and Observed Frequencies B. R. PARKER
Department of Physics
Idaho State University,
Pocafello,
Idaho 83202
AND
G. P. SHARE Department
of Miu-obiology
Idaho State University,
Pocatello,
Idaho 8%‘01
Theoretical absorption frequencies in DNA are compared with lines which occur in its infrared spectra. The comparison wa8 made in an effort to gain insight into the accuracy of theoretical hydrogen bond potential curves for DNA. The observed infrared lines do not correlate exactly with the theoretical predications. The reasons for this are discussed. I. INTRODUCTION
Several calculations have now been made of the potential experienced by the proton in the hydrogen bonds of DNA. Up to the present time there has been little or no comparison of these results to experiment. In the present paper an attempt is made to see which of these potential curves is in best agreement wit,h experiment. An accurate representation of this potential is important because the genetic code is contained in this region. Lowdin has suggested that mut,at#ions may result if the hydrogen bond proton shifts position within this potential (1). In his theory a permanent tautomeric rearrangement of proton pairs can occur via proton tunnelling. He assumes that each proton is locked in position in one well of a double well potential, and can tunnel through to t’he other well under the proper conditions. All original calculations indicated that the potent#ial wells were indeed double. The first calculatJions of the G-C potential were performed by Ladik (2); these were followed by more sophisticated quantum mechanical calculations by Rein and Harris (3). Lunell and Sperber then used Rein and Harris’ results to obt,ain several other potentials includning those of A-T and G-T (4). Recently, Clementi et al. (5)) have published a paper indicating that t,he G-C potential is not double, but rather it is single with a slight hump. If correct,, this could have serious consequences in Lowdin’s theory. Under the circumstances it was felt that, some kind of comparison to experiment was needed. 195 Copyright 0 1972 by .bzademic Press, Inc.
196
PARKER
AND
KHARE
CALF
$000
2000
3000
roll
c
POLV
u
POLV
a
POLV
I
POLV
G
WAVENUMBER
FIG. 1. Infrared spectra of the region of the predicted
It was hoped that a comparison of curring in the infrared spectra would best represented the actual potential. were obtained using the WKB method, cies of the hydrogen bond proton were given in Ref. (6).
TBVMUS-DOUBLE
CM
-1
absorptions.
predicted absorption lines with those octhrow some light on which of the curves The energy levels for each of the curves and from these the vibrational frequencalculated. Details of the calculation are
II. EXPERIMENTAL
The infrared was obtained on a Beckman IRSOA spectrophotometer with a spectral range from 4000 cm-’ to 250 cm-‘. Scan speeds of 5 min, 10 min, 24 min and 46 min were used on the various samples. Usually an initial 5 min scan was performed followed by one of either 24 or 46 min. Double stranded calf thymus and poly 1.~01~ C DNA along with single stranded poly I, C, U, G and A were used in the experiment’. Samples were made 1The poly I poly C double stranded and poly U, poly C, poly I and poly G single stranded were obtained from P. L. Biochemicals, Inc., Milwaukee, Wisconsin. The poly A and calf thymus were obtained from Mann Research Laboratories, New York. Calf thymus DNA was also obtained from Calbiochem, Los Angeles.
INFRARED
ABSORPTION TABLE
FREQUENCIES
197
IN DNA
I
A COMPARISON OF THEORETICXL FUNDAMENTAL ABSORPTION IN DNA CORRESPONDING EXPERIMENTAL ONES IN UNITS cm+ B Experiment
GCb
A-To
GCd
GCe
GC’
GCg
3200 2950 2800
2557
4138
4322
2476
3040
4864
8 All frequencies are based on energy level calculations using the WKB quencies in the first column are experimental, all others are theoretical. b Rein and Harris. c Lunell and Sperber. d Clementi et al.-HB 1 (hydrogen bond 1). e Clementi et al.-HB 2. f Clementi et al.-HB 3. g Rai and Ladik. ‘1 Rein and Harris.
TO THE
Gc excitedh
1516
method.
Fre-
up by mixing the DNA with KBr and pressing the mixture into pellets. Special precaut,ions were t,aken to make sure no water was absorbed in t’he sample. The KBr was stored in an oven at all t,imes when not’ in use, and t’he DNA was held under vacuum for several days before use. Several samples of DNA were also held at. a temperature of 40°C for several hours prior t.o t.he run to see if any difference could be detected. Little difference was noted, however, Finally, pellets of pure KBr were made up and checked for water. Their spectra was flat, indicat,ing little or no absorption of water. The infrared of double stranded DNA was compared for several cases to that resulting from single stranded DNA. In the first part of the experiment spectra n-as obtained from DNA in it.s usual double stranded form; in the second part. the DXA was denatured by heat t,o obtain single stranded DNA2. The spectra of t,he single stranded DNA (for the same sample) was then taken. Since hydrogen bonds are not intact in single DNA but are in DNA in its double helical form, the differences in the two spect’ra should be a result of the vibrations associated wit’h the protons in the hydrogen bond. These differences could be compared with the calculated frequencies. Initial results showed that t,he differences were small using these t,echniques; so commercially available double stranded I-C DNA was obtained along with the corresponding single stranded poly forms, poly I and * Calf thymus DNA dissolved in SSC (0.015 M NaCl and 0.015 M Na (Citrate) centration
of 0.1 mg/ml
in crushed ice-water ethanol;
bath. The single stranded
the precipitate
absorbance
at a con-
was heated in boiling water at 100°C for 12 min and rapidly DNA thus prepared was precipitated
was then dried at room temperature
was taken as an indication
for single strandedness
under vacuum. of the DNA
cooled by cold
An increase in preparation.
PARKER
19s poly C. Again t’he spectra now consider the results.
AND
of the double
III.
KHARE
and single forms were compared.
Let us
RESULTS
The region of the infrared that is of most interest in connection with the hydrogen bond potentials is that from 2500 cm-l to 4000 cm-‘. In this region there is, of course, the large broad overall hydrogen bond absorption line. We are nterested in the fine structure or small absorption lines associated with it. There appears to be absorpt8ions of this type at 3200 cm-l, 2950 cm-l, and 2SO0 cm-l (Fig. 1). They appear, however, in both t’he single and double forms of DNA. The line at 3200 cm-’ is usually considered to be a N-H stretching vibration. Note in our spectra that it is very strong in poly A, but particularly weak in poly U and poly I. This is consistent with the makeup of these forms. Poly A has three N-H stretch regions in a given base, while poly I and poly U only have one each. Poly A and poly C have two each. Table I shows the predicted resonant absorptions based on t’he various potentials. Rein and Harris’ potentials predict resonant absorptions at 2557 cm+ and 4135 cm+ for G-C and A-T, respectively. The 4138 cm-’ is outside the range of
POLV I ‘POLV
c
DOU6lE
4000
,000
WAVERUMBER
Ci’
4000
3000
WAVElUMBER
CV ’
FIG. 2. A comparison of the infrared spectra of single and double DNA. The upper two curves on t,he right were obtained using DNA from Mann labs. The lower two were obtained using DNA from Calbiochem.
INFRARED
ABSORPTION
FREQUENCIES
IN DNA
I!19
our spectrophotometer but, the 2557 cm-l is not. There does not, appear to be an absorpt,ion in the immediate neighborhood of 2557 cm-’ in the G-C spectra. The closest experimental absorptions are at 3800 cm-l and 2950 cm-‘. The three G--C potent,ial curves published by Clementi predict absorptions at 3476 cm-l, 3040 cm-l, and 4322 cm-‘. One of these, namely, 3040 cm-l, is close to the experiment:~I values. Also included in t.he t,able is the G-C excited pot’ential. This has been included
\.L .6
.6
LO
I.2
1.4 DISTANCE
1.6
1.8
FIG. 3. Potential curve of the hydrogen bond in guanine-cytosine and expected proton frequencies (R. Rein and F. E. Harris).
.5
.8
1.2
1.4 DISTANCE
2.0
2.2
ti,
1.6
1.8
showing energy levels
2.0
2.2
ti,
FIG. 4. Potential curve of the hydrogen bond in adenine-thymine and expected proton frequencies (S. Lunell and G. Sperber).
showing energy levels
PARKER
AND
KHARE
.5 _
.* _
1.5 -
1.0 _
I.5
1.0
1.5
2.0 DISTANCE
(i
2.5
1
FIG. 5. Potential curve of the hydrogen bond in guanine-cytosine showing energy levels and expected proton frequencies. (This is curve HB3 of E. Clementi et al.)
2.0
-
0.5 -
2.0
1.5 DISTAICE
2.0
2.5
Gil
FIG. 6. Potential curve of the hydrogen bond in guanine-cytosine showing energy levels and expected proton frequencies. (This is curve HB2 of E. Clementi et al.)
JNFRAREI)
ABSORPTION
FREQUENCIES
iv
IN DNA
201
I
FIG. 7. Regardless of whether the will become double as the bond pulls Since permanent tautomeric changes tunnelling will be important, even for
because there have been amount of time in excited mechanical pot,ential was taken directly from t$eir
e
hydrogen bond is double or single at equilibrium it apart. Diagrams A, B and C show how this happens. occur only during the unwinding process, quantllm a single well.
suggestions that the bases may spend a considerable states (7). Finally, Rein and Ladik’s G-C nonquantum also included. The energy levels for this potential were paper (8). IT’. 1)ISCUSSION
Table I indicates that there is some discrepancy between theory and experiment in all cases. Only one of t,he predicted absorptions (Clementi’s HB3) lies within the range of the broad experimental hydrogen bond absorption. It should be mentioned, however, that the problem has been treated in one dimension only. Actually it is a three-dimensional problem. All potentials discussed in this report are cross sections of the three-dimensional potential well along a line joining their centers. Stretching vibrations are assumed to take place along only this line. Also, vibrational coupling has not been taken into consideration. Coupling of this type might, for example, be possible between adjacent hydrogen bonds. It appears likely that some corrections of this type will have to be t,aken into consideration if theory and experiment are to be brought closer together. One of the difficulties in trying to identify an absorption which might be due to the proton in the hydrogen bond is distinguishing it, from the numerous other
PARKER
202
AND
KHARE
absorptions due to hydrogen which would be taking place in the same region of the spectra. It was hoped that by comparing double stranded DNA wit,h bonds intact to single stranded DNA with dangling bonds a difference would be noticed in this region. Slight shifts of various peaks have been noticed in other parts of the spectra under these circumstances. No distinct differences were not,iced in our experiment, however, in the region of hydrogen absorption. Despite this, absorptions due to the hydrogen bond proton should be visible. Finally, let us consider some of the consequences of Clement,i’s single well potential. If correct experimentally, it would appear to invalidate L&din’s mutation theory. The authors feel, however, that this is not the case. It should be noted that permanent tautomeric rearrangement of a base pair in DNA can take place only if the t’unnelling takes place within a very short time interval of the unwinding of the double helix. During part of this time interval the hydrogen bond is considerably stretched from its equilibrium (see Fig. 7) distance. This is, in fact, the most critical period for the protons-it is during this time t,hat they must decide whether to stay where they are or shift to positions across t,he well. Ladik has shown that the double well becomes much more pronounced during this time with the central hump increasing in height. Clementi also notes this in his calculations. His single well becomes double when the hydrogen bond is stretched. Since this is the time when permanent tautomeric rearrangement takes place it appears as if quantum tunnelling is important regardless of whether the well is double or single under equilibrium conditions It appears, moreover, that this building up of t’he pot,ential may be nature’s way of protecting against mutations at replication. When the bonds are intact and replication is not taking place mutations do not occur regardless of whether the well is double or single. ACKNOWLEDGMENTS This work was supported by grants from the Research Programs Committee, Idaho State University and the American Cancer Society, Idaho Division. Thanks are ext,ended to S. Lunell, F. R. Harris and E. Clementi for permission to reproduce potential curves.
RECEIVED: May 19, 1971 REFERENCES 1. P. 0. LOWDIN, Rev. Mod. Phys. 36, 724 (1963). d. J. L.IDIK, Preprint QBS, Quantum Chemistry Group, University of Uppsala, S. R. F. E. H.XRRIS, SND G. SPERBER, Preprint QB Quantum Chemistry Group,
REIN RAI
BND W. NIESSON, AND J. EVICRY, S. SVETINA, Int. J. Quantum J. L.~DIK, Mol. Spectrosc.
8,94
(1971).
Sweden.
OF
MOLECULAR
Infrared
SPECTROSCOPY
41, 195-202 (1972)
Spectra of DNA: Correlation of Theoretical and Observed Frequencies B. R. PARKER
Department of Physics
Idaho State University,
Pocafello,
Idaho 83202
AND
G. P. SHARE Department
of Miu-obiology
Idaho State University,
Pocatello,
Idaho 8%‘01
Theoretical absorption frequencies in DNA are compared with lines which occur in its infrared spectra. The comparison wa8 made in an effort to gain insight into the accuracy of theoretical hydrogen bond potential curves for DNA. The observed infrared lines do not correlate exactly with the theoretical predications. The reasons for this are discussed. I. INTRODUCTION
Several calculations have now been made of the potential experienced by the proton in the hydrogen bonds of DNA. Up to the present time there has been little or no comparison of these results to experiment. In the present paper an attempt is made to see which of these potential curves is in best agreement wit,h experiment. An accurate representation of this potential is important because the genetic code is contained in this region. Lowdin has suggested that mut,at#ions may result if the hydrogen bond proton shifts position within this potential (1). In his theory a permanent tautomeric rearrangement of proton pairs can occur via proton tunnelling. He assumes that each proton is locked in position in one well of a double well potential, and can tunnel through to t’he other well under the proper conditions. All original calculations indicated that the potent#ial wells were indeed double. The first calculatJions of the G-C potential were performed by Ladik (2); these were followed by more sophisticated quantum mechanical calculations by Rein and Harris (3). Lunell and Sperber then used Rein and Harris’ results to obt,ain several other potentials includning those of A-T and G-T (4). Recently, Clementi et al. (5)) have published a paper indicating that t,he G-C potential is not double, but rather it is single with a slight hump. If correct,, this could have serious consequences in Lowdin’s theory. Under the circumstances it was felt that, some kind of comparison to experiment was needed. 195 Copyright 0 1972 by .bzademic Press, Inc.
196
PARKER
AND
KHARE
CALF
$000
2000
3000
roll
c
POLV
u
POLV
a
POLV
I
POLV
G
WAVENUMBER
FIG. 1. Infrared spectra of the region of the predicted
It was hoped that a comparison of curring in the infrared spectra would best represented the actual potential. were obtained using the WKB method, cies of the hydrogen bond proton were given in Ref. (6).
TBVMUS-DOUBLE
CM
-1
absorptions.
predicted absorption lines with those octhrow some light on which of the curves The energy levels for each of the curves and from these the vibrational frequencalculated. Details of the calculation are
II. EXPERIMENTAL
The infrared was obtained on a Beckman IRSOA spectrophotometer with a spectral range from 4000 cm-’ to 250 cm-‘. Scan speeds of 5 min, 10 min, 24 min and 46 min were used on the various samples. Usually an initial 5 min scan was performed followed by one of either 24 or 46 min. Double stranded calf thymus and poly 1.~01~ C DNA along with single stranded poly I, C, U, G and A were used in the experiment’. Samples were made 1The poly I poly C double stranded and poly U, poly C, poly I and poly G single stranded were obtained from P. L. Biochemicals, Inc., Milwaukee, Wisconsin. The poly A and calf thymus were obtained from Mann Research Laboratories, New York. Calf thymus DNA was also obtained from Calbiochem, Los Angeles.
INFRARED
ABSORPTION TABLE
FREQUENCIES
197
IN DNA
I
A COMPARISON OF THEORETICXL FUNDAMENTAL ABSORPTION IN DNA CORRESPONDING EXPERIMENTAL ONES IN UNITS cm+ B Experiment
GCb
A-To
GCd
GCe
GC’
GCg
3200 2950 2800
2557
4138
4322
2476
3040
4864
8 All frequencies are based on energy level calculations using the WKB quencies in the first column are experimental, all others are theoretical. b Rein and Harris. c Lunell and Sperber. d Clementi et al.-HB 1 (hydrogen bond 1). e Clementi et al.-HB 2. f Clementi et al.-HB 3. g Rai and Ladik. ‘1 Rein and Harris.
TO THE
Gc excitedh
1516
method.
Fre-
up by mixing the DNA with KBr and pressing the mixture into pellets. Special precaut,ions were t,aken to make sure no water was absorbed in t’he sample. The KBr was stored in an oven at all t,imes when not’ in use, and t’he DNA was held under vacuum for several days before use. Several samples of DNA were also held at. a temperature of 40°C for several hours prior t.o t.he run to see if any difference could be detected. Little difference was noted, however, Finally, pellets of pure KBr were made up and checked for water. Their spectra was flat, indicat,ing little or no absorption of water. The infrared of double stranded DNA was compared for several cases to that resulting from single stranded DNA. In the first part of the experiment spectra n-as obtained from DNA in it.s usual double stranded form; in the second part. the DXA was denatured by heat t,o obtain single stranded DNA2. The spectra of t,he single stranded DNA (for the same sample) was then taken. Since hydrogen bonds are not intact in single DNA but are in DNA in its double helical form, the differences in the two spect’ra should be a result of the vibrations associated wit’h the protons in the hydrogen bond. These differences could be compared with the calculated frequencies. Initial results showed that t,he differences were small using these t,echniques; so commercially available double stranded I-C DNA was obtained along with the corresponding single stranded poly forms, poly I and * Calf thymus DNA dissolved in SSC (0.015 M NaCl and 0.015 M Na (Citrate) centration
of 0.1 mg/ml
in crushed ice-water ethanol;
bath. The single stranded
the precipitate
absorbance
at a con-
was heated in boiling water at 100°C for 12 min and rapidly DNA thus prepared was precipitated
was then dried at room temperature
was taken as an indication
for single strandedness
under vacuum. of the DNA
cooled by cold
An increase in preparation.
PARKER
19s poly C. Again t’he spectra now consider the results.
AND
of the double
III.
KHARE
and single forms were compared.
Let us
RESULTS
The region of the infrared that is of most interest in connection with the hydrogen bond potentials is that from 2500 cm-l to 4000 cm-‘. In this region there is, of course, the large broad overall hydrogen bond absorption line. We are nterested in the fine structure or small absorption lines associated with it. There appears to be absorpt8ions of this type at 3200 cm-l, 2950 cm-l, and 2SO0 cm-l (Fig. 1). They appear, however, in both t’he single and double forms of DNA. The line at 3200 cm-’ is usually considered to be a N-H stretching vibration. Note in our spectra that it is very strong in poly A, but particularly weak in poly U and poly I. This is consistent with the makeup of these forms. Poly A has three N-H stretch regions in a given base, while poly I and poly U only have one each. Poly A and poly C have two each. Table I shows the predicted resonant absorptions based on t’he various potentials. Rein and Harris’ potentials predict resonant absorptions at 2557 cm+ and 4135 cm+ for G-C and A-T, respectively. The 4138 cm-’ is outside the range of
POLV I ‘POLV
c
DOU6lE
4000
,000
WAVERUMBER
Ci’
4000
3000
WAVElUMBER
CV ’
FIG. 2. A comparison of the infrared spectra of single and double DNA. The upper two curves on t,he right were obtained using DNA from Mann labs. The lower two were obtained using DNA from Calbiochem.
INFRARED
ABSORPTION
FREQUENCIES
IN DNA
I!19
our spectrophotometer but, the 2557 cm-l is not. There does not, appear to be an absorpt,ion in the immediate neighborhood of 2557 cm-’ in the G-C spectra. The closest experimental absorptions are at 3800 cm-l and 2950 cm-‘. The three G--C potent,ial curves published by Clementi predict absorptions at 3476 cm-l, 3040 cm-l, and 4322 cm-‘. One of these, namely, 3040 cm-l, is close to the experiment:~I values. Also included in t.he t,able is the G-C excited pot’ential. This has been included
\.L .6
.6
LO
I.2
1.4 DISTANCE
1.6
1.8
FIG. 3. Potential curve of the hydrogen bond in guanine-cytosine and expected proton frequencies (R. Rein and F. E. Harris).
.5
.8
1.2
1.4 DISTANCE
2.0
2.2
ti,
1.6
1.8
showing energy levels
2.0
2.2
ti,
FIG. 4. Potential curve of the hydrogen bond in adenine-thymine and expected proton frequencies (S. Lunell and G. Sperber).
showing energy levels
PARKER
AND
KHARE
.5 _
.* _
1.5 -
1.0 _
I.5
1.0
1.5
2.0 DISTANCE
(i
2.5
1
FIG. 5. Potential curve of the hydrogen bond in guanine-cytosine showing energy levels and expected proton frequencies. (This is curve HB3 of E. Clementi et al.)
2.0
-
0.5 -
2.0
1.5 DISTAICE
2.0
2.5
Gil
FIG. 6. Potential curve of the hydrogen bond in guanine-cytosine showing energy levels and expected proton frequencies. (This is curve HB2 of E. Clementi et al.)
JNFRAREI)
ABSORPTION
FREQUENCIES
iv
IN DNA
201
I
FIG. 7. Regardless of whether the will become double as the bond pulls Since permanent tautomeric changes tunnelling will be important, even for
because there have been amount of time in excited mechanical pot,ential was taken directly from t$eir
e
hydrogen bond is double or single at equilibrium it apart. Diagrams A, B and C show how this happens. occur only during the unwinding process, quantllm a single well.
suggestions that the bases may spend a considerable states (7). Finally, Rein and Ladik’s G-C nonquantum also included. The energy levels for this potential were paper (8). IT’. 1)ISCUSSION
Table I indicates that there is some discrepancy between theory and experiment in all cases. Only one of t,he predicted absorptions (Clementi’s HB3) lies within the range of the broad experimental hydrogen bond absorption. It should be mentioned, however, that the problem has been treated in one dimension only. Actually it is a three-dimensional problem. All potentials discussed in this report are cross sections of the three-dimensional potential well along a line joining their centers. Stretching vibrations are assumed to take place along only this line. Also, vibrational coupling has not been taken into consideration. Coupling of this type might, for example, be possible between adjacent hydrogen bonds. It appears likely that some corrections of this type will have to be t,aken into consideration if theory and experiment are to be brought closer together. One of the difficulties in trying to identify an absorption which might be due to the proton in the hydrogen bond is distinguishing it, from the numerous other
PARKER
202
AND
KHARE
absorptions due to hydrogen which would be taking place in the same region of the spectra. It was hoped that by comparing double stranded DNA wit,h bonds intact to single stranded DNA with dangling bonds a difference would be noticed in this region. Slight shifts of various peaks have been noticed in other parts of the spectra under these circumstances. No distinct differences were not,iced in our experiment, however, in the region of hydrogen absorption. Despite this, absorptions due to the hydrogen bond proton should be visible. Finally, let us consider some of the consequences of Clement,i’s single well potential. If correct experimentally, it would appear to invalidate L&din’s mutation theory. The authors feel, however, that this is not the case. It should be noted that permanent tautomeric rearrangement of a base pair in DNA can take place only if the t’unnelling takes place within a very short time interval of the unwinding of the double helix. During part of this time interval the hydrogen bond is considerably stretched from its equilibrium (see Fig. 7) distance. This is, in fact, the most critical period for the protons-it is during this time t,hat they must decide whether to stay where they are or shift to positions across t,he well. Ladik has shown that the double well becomes much more pronounced during this time with the central hump increasing in height. Clementi also notes this in his calculations. His single well becomes double when the hydrogen bond is stretched. Since this is the time when permanent tautomeric rearrangement takes place it appears as if quantum tunnelling is important regardless of whether the well is double or single under equilibrium conditions It appears, moreover, that this building up of t’he pot,ential may be nature’s way of protecting against mutations at replication. When the bonds are intact and replication is not taking place mutations do not occur regardless of whether the well is double or single. ACKNOWLEDGMENTS This work was supported by grants from the Research Programs Committee, Idaho State University and the American Cancer Society, Idaho Division. Thanks are ext,ended to S. Lunell, F. R. Harris and E. Clementi for permission to reproduce potential curves.
RECEIVED: May 19, 1971 REFERENCES 1. P. 0. LOWDIN, Rev. Mod. Phys. 36, 724 (1963). d. J. L.IDIK, Preprint QBS, Quantum Chemistry Group, University of Uppsala, S. R. F. E. H.XRRIS, SND G. SPERBER, Preprint QB Quantum Chemistry Group,
REIN RAI
BND W. NIESSON, AND J. EVICRY, S. SVETINA, Int. J. Quantum J. L.~DIK, Mol. Spectrosc.
8,94
(1971).
Sweden.