Nucleic Acids Research Advance Access originally published online on May 21, 2007
Nucleic Acids Research 2007 35(Web Server issue):W713-W717; doi:10.1093/nar/gkm320
Nucleic Acids Research, 2007, Vol. 35, No. suppl_2 W713-W717
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
DSHIFT: a web server for predicting DNA chemical shifts
Sik Lok Lam*
Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
*To whom correspondence should be addressed. Tel: +852 2609 8126; Fax: +852 2603 5057; Email: lams{at}cuhk.edu.hk
Received January 27, 2007. Revised March 31, 2007. Accepted April 17, 2007.
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ABSTRACT
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DSHIFT is a web server for predicting chemical shifts of DNA
sequences in random coil form or double helical B-form. The
prediction methods are based on sets of published reference
chemical shift values and correction factors which account for
shielding or deshielding effects from neighboring nucleotides.
Proton, carbon and phosphorus chemical shift predictions are
available for random coil DNAs. For double helical B-DNA, only
proton chemical shift prediction is available. Results from
these predictions will be useful for facilitating NMR resonance
assignments and investigating structural features of solution
DNA molecules. The URL of this server is:
http://www.chem.cuhk.edu.hk/DSHIFT.
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INTRODUCTION
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Chemical shift contains a wealth of structural information of
DNAs. At present, several methods have been established to predict
chemical shifts of DNAs in random coil form (
1–3) and
double helical B-form (
4,
5). These methods are based on sets
of reference chemical shift values and correction factors from
experimental measurements, statistical analysis or semi-empirical
calculations. Shielding or deshielding contributions from nearest
neighbor and/or next-nearest neighbor nucleotides have been
included in these prediction methods. To automate these prediction
methods, a web server called ‘DSHIFT’ has been established
for predicting DNA chemical shifts in this work. This web server
is open access to everyone. Through entering a DNA sequence,
random coil or double helical B-DNA chemical shifts will be
predicted.
DSHIFT results can provide a quick reference guide for resonance assignments based on conventional NOESY and COSY-type experiments, thus facilitating solution structure studies of DNAs. These results can also provide useful information for studying structure–chemical shift relationship, identifying unstructured or right-handed double helical regions, monitoring DNA–drug or DNA–protein binding, and investigating conformational details of special features in DNA structures.
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WEB SERVER IMPLEMENTATION AND LAYOUT
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DSHIFT is written in HyperText Markup Language (HTML) and Active
Server Pages (ASP). Presently, it is implemented on Internet
Information Services (IIS) 6.0 using Microsoft Windows Server
2003 operating system. The web server is running on a Dell OptiPlex
GX270 Intel Pentium 4 2.4 GHz computer with 512 MB RAM and 80
GB hard drive.
Figure 1 shows the layout of DSHIFT, which is composed of basically three major types of pages, namely, ‘DSHIFT Home’, ‘Sequence Input’ and ‘Prediction Result’. ‘DSHIFT Home’ (Figure 2) is the starting page of DSHIFT which provides access to predict DNA chemical shifts in either random coil form or double helical B-form. ‘Sequence Input’ is the input page of DSHIFT. Sequence content, choices of nucleus and method will be submitted through this page. All input will be validated before submitting to prediction. If the input is valid, sequence information and chemical shift prediction results will appear in ‘Prediction Result’, which is the output page of DSHIFT. Finally, you have the options to either predict another sequence or go back to ‘DSHIFT Home’.
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SEQUENCE INPUT
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Figure 3A shows ‘Sequence Input’ page. You will
find the citations of method that you are going to use at the
beginning. For random coil chemical shift prediction, you have
to (i) enter the sequence and (ii) pick the nucleus that you
are interested in. Besides the choice of a particular nucleus,
there is also an option ‘Show ALL’ in case you want
to predict the chemical shift of all available nuclei. For double
helical B-DNA, in addition to the above input items, you have
to choose from ‘Altona method’ (
5) or ‘Wijmenga
method’ (
4) as shown in
Figure 4A. Due to method limitation,
chemical shift predictions of labile protons in double helical
B-DNA such as guanine imino (G-NH), thymine imino (T-NH), cytosine
bound amino (C-NHB) and free amino (C-NHE) are only available
in ‘Altona method’.
Sequence input has to be started from the 5'-end. Both upper
case and lower case characters of ‘C’, ‘G’,
‘A’ and ‘T’ are accepted. Space is also
allowed in sequence input. In case there is a typing mistake
in sequence or if nucleus or prediction method (in B-DNA) has
not been selected, an error message will appear, detailing the
input mistake. Due to serious spectral overlap and broadening
of resonance signals, DNA structures containing more than 100
nt are seldom studied by NMR spectroscopy. In DSHIFT, the maximum
length of sequence input has been set to 500 nt. An error message
will appear if the sequence length is longer than 500 nt.
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PREDICTION METHODS
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Random coil
In DSHIFT, random coil proton (1), carbon (2) and phosphorus
(3) DNA chemical shift predictions are available. For random
coil proton prediction, a pentamer model
which is based on a set of triplet chemical
shift values,
and next
nearest neighbor correction factors, has been used (
1), i.e.
 | (1) |
where
pred(X) is
the predicted chemical shift of X,
and
are the 5'- and
3'-next nearest thymine effects on X in the original 17-nt random
coil sequences,
and
are the real 5'- and 3'-next nearest effects
on X in the predicted sequence. Apart from the published data
set of H6/H8, H1', H2', H2'' and H3' which gives a prediction
accuracy of 0.02–0.03 p.p.m., adenine H2 (A-H2), cytosine
H5 (C-H5) and thymine H7 (T-H7) triplet values from the original
17-nt random coil sequences have also been extracted in this
work (Supplementary Data S1). Besides, next nearest neighbor
correction factors (Supplementary Data S2) have been derived
using the same strategy (
1) for these protons and the prediction
accuracy has been found to be 0.02 p.p.m.
Due to the absence of phosphate groups at the 5'- and 3'-termini, random coil proton chemical shifts for nucleotides at penultimate positions are calculated using the following equations:
 | (2) |
 | (3) |
where
and
are the 5'- and 3'-penultimate correction factors
(Supplementary Data S3), respectively.
For random coil carbon chemical shifts, the prediction method is based on a trimer model as only nearest neighbor effect has been found to be significant (2), i.e.
 | (4) |
The reported prediction accuracy of this
method is 0.09–0.10 p.p.m. Since the absence of phosphate
groups at both termini has been found to only affect the predicted
C3' values of the 3'-penultimate nucleotides (
2), therefore
3'-penultimate correction of C3' (Supplementary Data S3) has
also been included in this prediction method. In addition to
the published values of C6/C8, C1', C2' and C3', triplet values
of adenine C2 (A-C2), cytosine C5 (C-C5) and thymine C7 (T-C7)
have also been extracted from the original 17-nt random coil
sequences (Supplementary Data S1). The prediction accuracy for
these carbons has been found to be 0.03–0.04 p.p.m.
For phosphorus, prediction can be made either based on a trimer model (3), i.e.
 | (5) |
or a dimer model, i.e.
 | (6) |
In both cases, due to the absence of phosphate groups
at both 5'- and 3'-termini, end corrections to 5'- and 3'-penultimate
nucleotides have been included in these prediction methods.
In addition, end correction to 3'-terminal nucleotide is also
included in the dimer model. The reported phosphorus chemical
shift prediction accuracy is 0.02 and 0.03 p.p.m. for the trimer
and dimer models, respectively.
Double helical B-DNA
In DSHIFT, two prediction methods are available for double helical B-DNA. In Altona method, proton chemical shift prediction is based on a trimer model in which an incremental scheme and statistical reference values from experimental results have been used (5). The chemical shift of proton X, pred(X), in a given central residue X in a triplet N5'XN3', or at either terminal positions in a doublet E5'XN3' or N5'XE3' is predicted as:
 | (7) |
 | (8) |
 | (9) |
where X represents
the reference value, N
5' and N
3' represent the incremental shielding
or deshielding contributions of the four flanking residues on
the 5'- and 3'-side, respectively, E
5' and E
3' represent the
effect due to the absence of a phosphate group and attached
residue at the 5'- and 3'-termini, respectively. Deshielding
corrections have also been made to both 5'- and 3'-penultimate
nucleotides and the reported prediction accuracy of this method
is 0.01–0.03 p.p.m.
In Wijmenga method, proton chemical shift of a specific nucleotide is predicted based on a set of calculated reference shift value (ref) plus the chemical shift effect induced by its own base (ib), its 3'- (3'b) and 5'-neighboring bases (5'b) (4), i.e.
 | (10) |
These calculated
values are based on semi-empirical relations derived by Giessner-Prettre
and Pullman (
6) and the reported proton chemical shift prediction
accuracy of this method is 0.17 p.p.m.
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PREDICTION RESULT
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Figures 3B and
4B show examples of ‘Prediction Result’
pages of random coil and B-DNA, respectively, in which the sequence
input, length of sequence and a table summarizing the predicted
chemical shifts will be reported. The abbreviation, ‘n.a.’
(means not available), will be given to items that cannot be
predicted due to either absence of the nucleus or method limitation.
If the ‘Show ALL’ option has been selected instead
of a nucleus, prediction results of all available nuclei would
be summarized in the resulting table. At the end of this output
page, the total number of times that DSHIFT has been successfully
used will also be reported.
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SCOPE AND LIMITATIONS
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DSHIFT serves as a convenient tool for predicting chemical shifts
of DNAs in random coil form or double helical B-form. The predicted
values are referenced to the most upfield signal of 2,2-dimethyl-2-silapentane-5-sulfonate
sodium salt (DSS) and provides a quick reference guide which
facilitates the resonance assignment step in solution DNA structure
studies. The prediction accuracy of various methods adopted
in DSHIFT depends mainly on DNA conformations. Since temperature
and solution conditions affect stabilities of DNA structures,
it is expected that these factors will also affect the prediction
accuracy.
Temperature
For random coil chemical shift prediction, a large deviation between experimental and predicted values will be expected if residual structures are present in DNA sequences. Higher temperatures tend to eliminate residual structures. Since temperature coefficients of random coil chemical shifts have been found to be quite small and they are in the same order of magnitudes as the measurement uncertainties (1–3), the predicted values should agree well with experimental values measured over a wide temperature range.
For double helical B-DNA, higher temperatures lead to denaturation of double helix and thereby a change in DNA conformation. Thus, the prediction accuracy is expected to decrease when the experimental values are obtained at temperatures where the double helix becomes unstable. For prediction of G-NH and T-NH chemical shifts, the predicted values in Altona method have been normalized to values measured at 15°C. For values at other temperatures, corrections have to be made using the temperature coefficients -2.2 x 10–3 and –5.4 x 10–3 p.p.m./°C, respectively (5).
Solution condition
For random coil chemical shift prediction, the values have been tested to represent the fully denatured state (1). Apart from temperature, pH, salt content and whether denaturant is present in solution will also affect the denatured state. If any of these factors favors the presence of residual structures, the predicted values will be expected to deviate greatly from the experimental results.
For double helical B-DNA predication, Altona and co-workers have found that the variations of monovalent salt concentration from 5 to 200 mM or divalent salt concentration from 0 to 20 mM in DNA samples do not lead to significant chemical shift changes (5). Nevertheless, pH and salt content affect the stability of B-form structures. Large deviations between the predicted and experimental values will be expected if pH or salt content does not favor the formation of stable B-DNA structure.
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SUPPLEMENTARY DATA
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Supplementary Data are available at NAR Online.
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ACKNOWLEDGEMENTS
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I would like to thank the valuable input and suggestions from
my students C. K. Kwok, K. F. Lai and L. M. Chi. I would also
like to thank K. F. Woo and D. Fenn for setting up the hardware
and software for this web server and all people who have tested
this server. The work described in this article was partially
supported by a grant from the Research Grants Council of the
Hong Kong Special Administrative Region (Project No.: 400704)
and a direct grant from CUHK Research Committee Funding (Project
No.: 2060252). Funding to pay the Open Access publication charge
was provided by Research Grants Council of the Hong Kong Special
Administrative Region.
Conflict of interest statement. None declared.
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REFERENCES
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- Lam SL, Ip LN, Cui X, Ho CN. Random coil proton chemical shifts of deoxyribonucleic acids. J. Biomol. NMR (2002) 24:329–337.[CrossRef][Web of Science][Medline]
- Kwok CW, Ho CN, Chi LM, Lam SL. Random coil carbon chemical shifts of deoxyribonucleic acids. J. Magn. Reson (2004) 166:11–18.[CrossRef][Web of Science][Medline]
- Ho CN, Lam SL. Random coil phosphorus chemical shift of deoxyribonucleic acids. J. Magn. Reson (2004) 171:193–200.[CrossRef][Web of Science][Medline]
- Wijmenga SS, Kruithof M, Hilbers CW. Analysis of 1H chemical shifts in DNA: assessment of the reliability of 1H chemical shift calculations for use in structure refinement. J. Biomol. NMR (1997) 10:337–350.[CrossRef][Web of Science]
- Altona C, Faber DH, Westra Hoekzema AJA. Double-helical DNA 1H chemical shifts: an accurate and balanced predictive empirical scheme. Magn. Reson. Chem (2000) 38:95–107.[CrossRef][Web of Science]
- Giessner-Prettre C, Pullman B. Quantum mechanical calculations of NMR chemical shifts in nucleic acids. Q. Rev. Biophys (1987) 20:113–172.[Web of Science][Medline]
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