Alexander M. Andrianov
Institute of Bioorganic Chemistry,
Belarus Academy of Sciences,
Kuprevich St., 5/2,
220141 Minsk, Republic of Belarus
The high mutability of HIV envelope proteins is a major obstacle to creating efficient means of AIDS prevention. Therefore much effort is now focused on revealing conserved regions in HIV proteins and establishing their role. In particular, the HIV-1 principal neutralizing determinant (PND; V3 loop Cys303-Cys338 in gp120), albeit hypervariable in general, is clearly conserved in the Gly-Pro-Gly-Arg-Ala-Phe stretch, the most probable viral immunodominant epitope.
To better understand the effect of sequence variation on the conformation and antigenicity of the HIV-V3 loop, we determined the local structures of the HIV-1 PND from the MN, RF, Haitian, and Thailand isolates in water and in water/TFE mixed solvent and compared these conformations among themselves by the statistical criteria. A probabilistic approach (1) realized in the computer program CONFNOE is employed to calculate the main chain conformations of the HIV-1 V3 loops, based on the published sequentia l d connectivity data (2-4).
In four forms of the HIV-1 V3 loops, the conformational states of all amino acid residues are determined, the secondary structure established, and the conformationally rigid and mobile regions identified. The following major conclusions are made based on the comparative analysis of the simulated PND conformations: i) in spite of sequence variation, there are conserved secondary structural elements inside the V3 loops; ii) for separate amino acid residues, the conformational differences between their local structures in water and in water/TFE mixed solvent are found to be statistically significant; iii) no ordered helix is induced at the C-terminal site of the HIV-1 PND by the addition of trifluoroethanol; iv) the crest of the V3 loop containing the Gly-Pro-Gly (Arg, Lys, Gln) adopts a beta-turn conformation, which may be the basis of its conservation in many HIV-1 isolates.
The results obtained are completely in line with the d-connectivity data (2-4) used to calculate the most probable conformations of the HIV-1 PND and, despite the similarity of the main conclusions, are characterized by a number of substantial differences from those of the computer analysis reported earlier (2-4).
References and Footnotes
1. S.A. Sherman, A.M. Andrianov and A.A. Akhrem, J.
Biomol. Struct. Dynam. 4, 869-884 (1987)
2. P. Catasti, J.D. Fontenot, E.M. Bradbury and G. Gupta, J. Biol. Chem.
270, 2224-2232 (1995)
3. P. Catasti, E.M. Bradbury and G. Gupta, J. Biol. Chem. 271, 8236-8242
(1996)
4. G. Gupta, G.M. Anantharamaiah, D.R. Scott, J.H. Eldridge and G. Myers,
J. Biomol. Struct. Dynam. 11, 345-366 (1993)
G. Bischoff*, U. Gromann, S. Lindau, R. Skölziger, W. Witkowski,
C. Bohley, W.-V. Meister and S. Hoffmann
Martin Luther University Halle-Wittenberg,
Department of Biochemistry,
Kurt-Mothes-Str. 3,
D-06120 Halle (Saale), Germany
*Author to whom correspondence should be addressed. E-mail: bischoff@biochemtech.uni-halle.de;
http://www.chemie.fu- berlin.de/_bischoff
Several 2,7-bis-[(dialkylamino)-acetamido]-fluoren-9-one derivatives were synthesized [1,2] as analogues of the DNA binding compound tilorone (2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one) and examined by their binding to DNA. The DNA binding characteristics were evaluated using UV and circular dichroism studies. Like tilorone, the fluorenone derivatives also intercalate through the bases resulting in increased Tm values and induced CD signals. A preference to alternated A-T and G-C sequences could be detected while no interaction to homologous sequences could be observed [3]. Moreover, no limit in the DNA-drug ratio was found, tested until the precipitation limit. These results are connected with our findings examined by surface plasmon resonance in the case of tilorone and 2,7-bis-[(dipropylamino)-acetamido]-fluoren-9- one [4], where an astonishing drug/base-pair ratio could be observed, indicative of a multitude of interaction facilities.
Molecular modeling calculations, by optimizing the geometry of the complexes under the assumption of intercalative and multitude suprahelical arrangements, exemplarily illustrate the molecular situation.
In accompanied observations, the pharmacological significance of the drugs was found in cytokine inducing and reverse transcriptase inhibiting efficiencies. Based on the thermodynamic and biological studies, a structure-function model is proposed.
References and Footnotes
1. S. Hoffmann, W. Witkowski, G. Borrmann, Z. Chem.
17 (1977) 291-293
2. S. Hoffmann, R. Skölziger, W. Witkowski, Z. Chem. 25 (1985)
432-433
3. W. Witkowsi, S. Hoffmann, A. Veckenstedt, G. Luck, Ch. Zimmer, R. Skölziger,
Acta biol. med. germ. 38 (1979) 733-737
4. G. Bischoff, R. Bischoff, U. Gromann, E. Birch-Hirschfeld, W.-V. Meister,
S. de A. Bambirra, S. Hoffmann, J. Biomol. Struc. Dyn. 16 (1998)
187-203
S. Vaitheeswaran1, R. D. Blake1, C. E. Tarr1 and K. D.
Bishop2
1Department of Physics and Astronomy,
5709 Bennett Hall,
University of Maine,
Orono, ME 04469-5709
Phone: 207-581-1038; Fax: 207-581-3410
2Department of Chemistry,
Michigan State University,
East Lansing, MI 48824
1- and 2-D proton NMR and T1 relaxation data have been used to study the interactions of formamide and 5'-GMP. The sites at which this commonly used denaturant preferentially binds to the nucleotide have been identified. This investigation forms the basis of similar studies of interactions with model oligomers to determine the mechanisms of denaturation of DNA by formamide.
References and Footnotes
1. Blake, R. D., Delcourt, S. G, Nucleic Acids Res.
24, 2095-2103 (1996).
Louis Carlacci
Department of Chemistry, CHE 305,
University of South Florida,
Tampa, Florida 33620-5250
*For author correspondence. Phone: (813)974-9622; Fax: (813)974-1733; E-mail:
lou@finch.cas.usf.edu
Conformational searches for the lowest total energy loop conformations
are computed by multiple independent Monte Carlo simulated annealing runs
starting from random initial conformations. The function optimized consists
of Lennard Jones nonbonded and H-bond potentials, coulomb and torsion potentials
and solvation free energy based on solvent accessible surface. Calculations
of 5- 7- and 9-residue loops in bovine pancreatic trypsin inhibitor (BPTI)
are more efficient. The methodology employed expands on approach used in
J. Comp. Chem., V. 17, 1996, pp. 1002-1012), which may be called a smart
algorithm approach to conformational searches. Knowledge based on protein
x-ray crystal structures, protein folding, and previous experience with
peptide conformational searches are used to narrow the search and/or make
it more realistic. The 9-residue loop interacts with two buried water molecules.
A 12-reisdue loop that interacts with 4 buried water molecules is also computed.However,
the total energy of the computed x-ray conformation is significantly greater
that the energy of the lowest energy conformation. The question that needs
to be answered is: Is the problem with the 12-residue loop calculation due
to the limitations of the conformational search or due to the thermodynamics
of the buried water molecules? If the latter is the case, the results of
this study should be useful in the development of methodologies that consider
water explicitly during the conformational search.
Feng Sha and Fu-Ming Chen*
Department of Chemistry,
Tennessee State University,
Nashville, TN 37209-1561
*Author to whom correspondence should be addressed. Phone: (615)963-5325;
Fax: (615)963-5434; E-mail: chenf@harpo.tnstate.edu.
Earlier calorimetric studies by others had indicated that despite the
absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds
strongly to actinomycin D with high cooperativity and a 2:1 drug to duplex
ratio. A subsequent optical spectral study with related oligomers from this
laboratory further suggested that ACTD may in fact stack on the G?C base
pairs at both ends of this oligomeric duplex. New findings are reported
herein to indicate that despite the lack of complete self-complementarity,
oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD
binding affinities with binding constants of roughly 2 x 107 M-1 and binding
densities of 1 drug molecule per strand (or 2 drugs per duplex). ACTD binding
affinity for the corresponding hetero-duplex obtained by annealing these
two oligomers is considerably reduced. Base replacement or removal of dC
and dG from the respective ends results in more than an order of magnitude
reduction in the ACTD binding strength, whereas appending base(s) at sequence
terminal(s) results in somewhat reduced binding affinities. It was further
found that despite the presence of additional XCG unit, the 11-mers d(CGXCGXCGXCG)
exhibit nearly identical binding affinities and binding densities as those
of the parent octamers. All these results strongly support the notion that
ACTD binds to d(CGXCGXCG)2 via stacking at the G?C base pairs of the sequence
terminals and the presence of proximal X/X mismatches appears to facilitate
the snug fits of the pentapeptide rings in the minor groove.
David Fushman, R.Xu and David Cowburn*
The Rockefeller University,
1230 York Avenue,
New York, New York 10021
*To whom the correspondence should be addressed. Phone: 212 327-8270; Fax:
212 327-7566; E-mail: cowburn@rockefeller.edu
Current methods of high-resolution structure determination are generally applicable to macromolecular assemblies formed by tight contacts between the individual, well-structured components. These methods have more limited applicability in those cases where there are weaker interactions between the constituents; examples include the relatively transient associations formed in complexes involved in signal transduction, or in transcriptional control. Crystal structures of such complexes might be biased by packing forces comparable to the interdomain interactions, while the precision and accuracy of the conventional NMR structural approaches are necessarily limited by the restricted number of NOE contacts and by interdomain flexibility rendering the available NOE information uninterpretable. The NMR relaxation approach [1-5] which takes advantage of the anisotropic character of the overall rotation, allows a direct determination of axes of hydrodynamic motion in solution. This approach is applied here to determine the change of domain orientation between unligated and dual ligated subdomains of the SH(32) fragment of Abelson kinase. These results demonstrate that direct analysis of interdomain orientations and motions of proteins in solution can be obtained from NMR relaxation analysis. This provides a method of describing changes in macromolecular assemblies not available by other methods and generally applicable to a wide range of macromolecules in their native milieu.
Supported by NIH GM-47021
References and Footnotes
1. Bruschweiler, R., Liao, X., Wright, P. E., Science
268, 886-9 (1995)
2. Tjandra, N.; Feller, S. E.; Pastor, R. W.; Bax, A., J. Am. Chem. Soc.
117, 12562-6 (1995)
3. Lee, L. K., Rance, M., Chazin, W. J. & Palmer, A. G., III., J.
Biomol. NMR 9, 287-98 (1997) 4. Copie, V., Tomita, Y., Akiyama, S. K.,
Aota, S., Yamada, K. M., Venable, R. M., Pastor, R. W., Krueger, S. &
Torchia, D. A., J. Mol. Biol. 277, 663-682 (1998)
5. McDonnell, J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J., Cowburn
D., Cell 96, XXX, (1999).
L. David Finger and Juli Feigon
Department of Chemistry and Biochemistry,
University of California,
Los Angeles, CA 90095-1569
Isolation of RNA sequences capable of binding biological cofactors in vitro provides support for the RNA world hypothesis (1). Benner et. al.have proposed that RNA could have catalyzed its own conversion to DNA in the pre-biotic world with assistance from the biological cofactor, cyanocobalamin (vitamin B12) (2). If this hypothesis is correct, RNA sequences which are capable of forming a complex with vitamin B12 should exist. Lorsch and Szostak have reported the in vitro selection of RNA aptamers which specifically bind vitamin B12 (3). The cyanocobalamin aptamer consensus sequence consists of 31 nucleotides which are predicted to form an unusual H-type pseudoknot (4). Biochemical characterization showed that the cyanocobalamin aptamer displays one of the highest affinities for a small molecule ligand (KD90nM). Furthermore,the aptamers affinity for the ligand is extremely dependent on the presence of high concentrations of Li counterions (1M). Decreasing the [Li+] drastically reduces the aptamers affinity for the ligand. In addition, substitution of 1M Na+, 1M K+, or 1M Na+/5mM Mg2+ for Li+ abolishes binding completely (3). Therefore, structural studies of the cyanocobalamin aptamer may provide confirmation for the existence of this unusual H-type pseudoknot and a rationale for its high ligand affinity and strict counterion dependence.
We are investigating the interaction of vitamin B12 with several different consensus aptamers from the selection pool using NMR. In the absence of cyanocobalamin, imino proton resonances from only a few base pairs in putative stem regions are observed. Upon the addition of cyanocobalamin,large changes in the imino proton spectra are observed and several new imino proton resonances appear, consistent with binding of vitamin B12 and folding of the aptamer. Results on the effects of Li+ concentration, pH, and RNA sequence on the folding of the aptamer in the presence of cyanocobalamin will be presented.
References and Footnotes
1. Szostak, J.W., Ellington, A.D., In Vitro Selection
of Functional RNA Sequences in The RNA World, Cold Spring Harbor Laboratory
Press, 511-533 (1993).
2. Benner, S.A., Ellington, A.D., Traver, A., Proc. Natl. Acad. Sci.
U.S.A. 86, 7054-7058 (1989).
3. Lorsch, J.R., Szostak, J.W., Biochemistry 33, 973-982 (1994).
4. Pleij, C.W.A., Appendix 2:RNA Pseudoknots in The RNA World, Cold
Spring Harbor Laboratory Press, 609-613 (1993).
David Fushman*1, Nico Tjandra2
and David Cowburn1
1The Rockefeller University,
1230 York Avenue,
New York, New York 10021
2The Laboratory of Biophysical Chemistry,
National Heart, Lung, and Blood Institute,
National Institutes of Health,
Bethesda, Maryland 20892-0380
*Author to whom correspondence should be addressed. Phone: 212 327-8274;
Fax: 212 327-7566; E-mail: fushmad@rockefeller.edu
NMR relaxation methods are capable of providing information on protein dynamics, invaluable for understanding molecular mechanisms of protein folding, molecular recognition and catalysis. Current approaches to obtaining information on protein dynamics from 15N relaxation data are based on either model-free approaches (1) or the mapping of spectral density functions (2) and assume a constant value for the 15N chemical shift anisotropy (CSA), as inferred from solid state NMR data on short peptides. Recent analysis of multiple-field 15N relaxation and CSA/dipolar cross-correlation data for human ubiquitin (3) has indicated a range of 15N CSA values significantly larger than anticipated. These site-specific variations in CSA render inaccurate those analyses based on the assumption of a uniform single value of CSA. Obtaining accurate information on protein dynamics from 15N relaxation data requires new, robust approaches to data collection and analysis, free from assumptions about the 15N CSA and conformational exchange contributions to 15N linewidth.
Here we describe such an approach to the direct, residue-specific, determination of protein dynamics from 15N relaxation data, using R1, R2, and 15N{1H} NOE, at various fields. Using this approach, the spectral densities, the values of 15N CSA, and the conformational exchange contribution to 15N transverse relaxation are directly determined from the relaxation data for human ubiquitin. Using an anisotropic overall rotational diffusion model, the spectral densities are analyzed in terms of the order parameter and the correlation time of local motion. The residue-specific values of 15N CSA and the spectral densities obtained using this approach are in good agreement with those derived previously (3) from CSA/dipolar cross-correlation analysis.
Supported by NIH GM-47021
References and Footnotes
1. Lipari, G.; Szabo, A., J. Amer. Chem. Soc. 104,
4559-4570 (1982); Clore, et al., J.Amer.Chem.Soc. 112, 4989-4936 (1990)
2. Peng, J.; Wagner, G., J. Magn. Reson. 94, 82-100 (1992)
3. Fushman, D., Tjandra, N., Cowburn D., J. Am. Chem. Soc. 120, 10947-10952
(1998)
Michael K. Gilson1*, Laurent David1, Martha S. Head2, Ray Luo1, Kristy Mardis1, Michael
J. Potter1
1Center for Advanced Research in Biotechnology,
9600 Gudelsky Drive,
Rockville, MD, 20850
2Current address:
SmithKline Beecham,
UW 2430,
709 Swedeland Road,
King of Prussia, PA 19406
*Author to whom correspondence should be addressed. E-mail: gilson@carb.nist.gov
Most existing methods of computing binding free energies are either detailed but time-consuming free energy simulations, or fast, simplified energy component models. Between these two extremes, however, lies a broad middle ground with ample room for models of intermediate complexity that might retain much of the accuracy of the detailed approaches, yet that might be fast enough for day-to-day use in a molecular design project. A new class of computer models that occupies this middle ground is emerging from research in several laboratories. These ``predominant-states'' models compute binding affinities based upon the low energy conformations of the complex and of the free molecules, and the use of implicit solvent models makes the calculations efficient. I will discuss algorithms that implement the predominant states concept, and will present applications to several molecular systems for which experimental data are available.
References and Footnotes
1. Gilson, M. K., Given, J. A. and Head, M. S., A new class
of models for computing receptor-ligand binding affinities. Chem. and
Biol. 4:87-92, 1997.
2. David, L., Luo, R., Head, M. S. and Gilson, M. K., Computational study
of KNI-272, a potent inhibitor of HIV-1 protease. On the mechanism of preorganization.,
J. Phys. Chem. 103:1031-1044, 1999.
3. Luo, R., David, L., Hung, H., Devaney, J., and Gilson, M. K., The strength
of solvent-exposed salt bridges., J. Phys. Chem. 103: 727-736, 1999.
4. Luo,R., David,L., Head,M.S., Given,J.A. and Gilson,M.K. Nucleic acid
base-pairing and N-methylacetamide self-association in chloroform: Affinity
and Conformation. Biophys. Chem., in press.
Kevin M. Guckian, Thomas R. Krugh and Eric T. Kool
Department of Chemistry,
University of Rochester,
Rochester NY, 14627
The Klenow fragment of DNA polymerase I was recently found to synthesize
a nonpolar base pair with high efficiency despite the base pair's inability
to form hydrogen bonds. This base pair consists of an isosteric nonpolar
adenine analog, 4-methylbenzimidazole, and a isosteric nonpolar thymine
analog, difluorotoluene. We have now examined the solution structure of
the F-Z pair in the context of a 12 base pair DNA duplex. We find that despite
the destabilization caused by these substitutions, the F-Z pair adopts a
geometry similar to that of a T-A pair in the same context. The results
lend support to the importance of shape matching in replication.
W.-V. Meister1*, S. Lindau1,
A.L. Hauser2, C. Bohley1,
U. Gromann1, M. Madre3,
L. Kovalenko3, G. Bischoff1,
R. Zhuk3 and S. Hoffmann1
1Martin Luther University Halle-Wittenberg,
Institute of Biochemistry,
Kurt-Mothes-Str. 3,
D-06120 Halle (Saale), Germany
2Institute of Physical Chemistry,
Mühl- pforte 1,
D-06108 Halle (Saale), Germany;
3Institute of Organic Synthesis,
Aizkraukles 21,
LV-1006 Riga, Latvia
*Author to whom correspondnece should be addressed. Phone: ++49-345- 5524855,
Fax: ++49-345-5527011; E-mail: meister@biochemtech.uni- halle.de
During the cell cycle nucleic acids undergo various alternate condensation and decondensation processes. The local nucleic acid concentration remains high and the highly charged anionic polyelectrolyte molecules form in dependence on their concentration multiple liquid crystalline phases and microphases in aqueous solution. Up to now much remains to be understood about the organization and functional processes of nucleic acid molecules in the cell.
Here we report about optical, calorimetric and microscopic investigations of lyotropic nucleic acid matrix systems. Mesogenic behaviour of binary mixtures prepared from amphiphilic nucleobase derivatives - like S- methoxyethyl-L-cysteine modified adenine and hypoxanthine, 9-vinyladenine and 1-vinylcytosine as well as adenosine - and polynucleotide matrices is observed at concentrations higher than 50mg/ml under native conditions. Two- and three-stranded adenine- uracil, hypoxanthine-cytosine and adenine-hypoxanthine complexes are used for selfassembly studies. These organization processes are mainly attributed to the horizontal Watson/Crick- and Hoogsteen-hydrogen bondings as well as vertical stacking arrangements. Atomic force microscopy studies on the graphit wafer showing domains of highly ordered sample strands and computer modeling support our findings of nucleic acid organization.
References and Footnotes
1. S. Hoffmann and W. Witkowski, in: Mesomorphic Order
in Polymers and Polymerization in Liquid Crystalline Media (A. Blumstein,
ed.), Am. Chem. Soc. Symp. Ser. 74, 178-236 (1978)
2. S. Hoffmann, in Handbook of Liquid Crystal (D. Demus, J. Goodby,
G. W. Gray, H.-W. Spiess and V. Vill, eds.), Wiley-VCH, Weinheim , Vol.
3, Chap. VIII, pp. 393-452 (1998) (and references cited therein)
3. W.-V. Meister, A.-M. Ladhoff, S.I. Kargov, G. Burckhardt, G. Luck, S.
Hoffmann, Z. Chem. 30, 213-215 (1990)
K. Wojtuszewski1, M. Hawkins2 and I. Mukerji1
1Department of Molecular Biology and Biochemistry,
Molecular Biophysics Program,
Wesleyan University, Middletown, CT 06457
2Pediatric Branch,
NCI,
Bethesda, MD 20892
HU is a multifunctional DNA-binding protein implicated as an accessory
protein in a number of genomic events. These events include replication,
transcription, recombination and packaging of DNA. HU binds to DNA non-specifically
and gel mobility shift assays are suggestive that HU forms multimeric complexes
with DNA. These assays of multimeric HU-DNA complexes are indicative that
one HU molecule binds to approximately nine base pairs, consistent with
the HU-to-DNA ratio previously determined by DNA footprinting experiments.
Our research utilizes fluorescence anisotropy to determine quantitatively
the equilibrium binding constants of single and multiple HU dimers bound
to DNA in solution. The fluorescence anisotropy of HU complexed to DNA has
been monitored using a fluorescent guanosine analog, 3-methyl isoxanthopterin
(3-MI) incorporated into 13-, and 34- oligonucleotide length segments. Fluorescence
anisotropy measurements over a range of HU concentrations have been used
to determine the binding constant for both single and multiple species of
HU bound to DNA. These equilibrium binding constants are compared with those
determined by gel mobility shift assays.
Xiang-Jun Lu and Wilma K. Olson*
Department of Chemistry
Rutger, the State University of New Jersey
Wright-Rieman Laboratories
610 Taylor Road
Piscataway, NJ 08854-8087
*Author to whom correspondence should be addressed. Phone: (732) 445-3993;
Fax: (732) 445-5958; Email: olson@rutchem.rutger.edu
We present a rigorous comparison among seven of the most popular programs -- CEHS, CompDNA, Curves, FREEHELIX, NGEOM, NUPARM, RNA -- for analyzing helical nucleic acid structures. Our findings demonstrate that the choice of mathematics has only a limited effect on the computed parameters, even in highly deformed duplexes. The results are much more sensitive to the choice of reference frames. The disparate schemes yield very similar conformational descriptions if the calculations are based on a common reference frame. It follows then that:
(1) Any factor which influences the reference frame, such as fitting or not fitting standard bases to experimentally derived coordinates, will have a noticeable effect on both complementary base pair and dimer step parameters.
(2) The base pair parameters agree with each other better than the dimer step parameters because the distortions of two complementary bases can sometimes have a significant influence on the dimer step parameters.
(3) Specifically, Twist is sensitive to the difference of Shear (d-Shear), Shift to d-Opening, Tilt to d-Stagger and Rise to d-Buckle. Slide and Roll are much less effected.
References and Footnotes
1. X.J. Lu and W.K. Olson, J. Mol. Biol. 285 (4),
1563-1575 (1999).
2. X.J. Lu, M.S. Babcock and W.K. Olson, J. Biomol. Struct. Dynam.
(in the press) (1999).
Michael A. Livshits, Olga F. Borisova, Olga K. Mamaeva and Anna K.
Shchyolkina
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences,
117984, Moscow, Russia
Complexes formed by linked oligonucleotides 3'-d(GT)5-L-d(GT)5-3' (with two kinds of non-nucleotide linkers L1=pO(CH2)6; L2=pO(CH2CH2O)3p) were studied by UV absorption, CD and fluorescence spectros copy. Ethidium bromide polarized fluorescence was used for determination of relaxation times and hydrodynamic volumes of the complexes. The decay times of heterogeneous fluorescence of acridine orange served for evaluation of the fractions of hydrogen-bonded and unpaired nucleotides. Bimolecular complexes taking a parallel four-stranded structure with five stacked G-quartets and with unpaired (buldged out) thymines were identified. Thermodynamic parameters of the complexes determined by fitting UV- and CD-melting curves as well as by the oligonucleotide concentration dependence of melting point proved to be in reasonable agreement. The hydrophobicity difference between linkers L1, L2 did not influence the quadruplex thermodynamics. Ions were shown to stabilize GT-qudruplexes predominantly entropically in the order: Mg++>Na+=K+. Obtained enthalpy values per G-quartet in the novel GT-quadruplexes (H=5.5 +- 0.5 kcal/mol) were found to be considerably lower than those in well known G-quadruplexes formed by parallel Gn strands. The modest energetics of the GT-quadruplexes seems to correspond to their supposed structural lability contrasting to very high rigidity of "continuous" G-quadruplexes.
Supported by RFBR grants 96-15-98093 ("Leading Scientific Schools")
and 96-04-50703
Nancy Stellwagen*
Department of Biochemistry,
University of Iowa,
Iowa City, IA 52242
*For author correspondence. E-mail: stellwag@blue.weeg.uiowa.edu
Recently, the free solution mobility of DNA was measured by capillary
electrophoresis in three different buffers, Tris-acetate-EDTA (TAE), Tris-borate-EDTA
(TBE), and isoelectric histidine buffer. In TAE buffer, the electrophoretic
mobility of high molecular weight DNA was approximately equal to the mobility
observed by classical Tiselius methods in NaCl solutions of comparable conductivity.
Hence, Tris ions and sodium ions either do not interact specifically with
DNA or interact in a comparable manner. However, the electrophoretic mobility
observed in TBE buffer was ~20% higher than observed in TAE buffer, due
to the formation of highly charged DNA-borate complexes in TBE buffer. Similar
DNA-borate complexes are observed during polyacrylamide gel electrophoresis,
but not in agarose gels, because of the competition of the agarose gel fibers
for the borate residues. DNA-buffer complexes are also present in isoelectric
histidine buffers. The complexes are large and appear to be stabilized by
electrostatic interactions, because they can be disrupted by adding neutral
salts to the solution. The combined results indicate that the buffers in
which DNA is studied cannot automatically be assumed to be innocuous.
Loren Dean Williams*, Lori McFail-Isom and Chad C. Sines
School of Chemistry & Biochemistry,
Georgia Institute of Technology,
Atlanta, GA 30332-0400
*Author to whom correspondence should be addressed. Phone: (404) 894-9752;
E-mail: loren.williams@chemistry.gatech.edu
Effects of salt on bending of AT-tracts (1-3) and GC-tracts (4-6) demand a central role for electrostatics in bending and other deformations. Yet most analysis of DNA structure, originating with "Calladine's Rules", has employed a base-base paradigm. This mind-set focuses on direct base-base interactions, discounting electrostatic contributions. Limitations on electrostatic analysis are partially technical; one simply cannot find many cations in low and medium resolution x-ray structures. These limitations explain the durability of the base-base paradigm in spite of obvious deficiencies. We that believe useful predictive models of both DNA and RNA structures will ultimately depend on explicit incorporation of electrostatic interactions.
Localization of cations in x-ray structures is therefore a critical step in structure determination. We have observed monovalent cations in the minor groove of AT-tacts, especially at a six coordinate solvent binding site at ApT steps. These peaks of cation density suggest that cation density is depleted elsewhere. In sum it is reasonable to propose a model in which nucleic acids are deformed by uneven cation density within the counterion atmosphere. The deformation can be global, as in axial bending, and local, as in groove width variation.
References and Footnotes
1. Diekmann, S. and Wang, J. C. (1985) J. Mol. Biol.
186, 1-11.
2. Laundon, C. H. and Griffith, J. D. (1987) Biochemistry 26, 3759-3762.
3. Shlyakhtenko, L. S., Lybubchenko, Y. L., Chernov, B. K. and Zhurkin,
V. B. (1990) Mol. Biol. (Moscow) 24, 79-95.
4. Brukner, I., Susic, S., Dlakic, M., Savic, A. and Pongor, S. (1994) J.
Mol. Biol. 236, 26-32.
5. Dlakic, M. and Harrington, R. E. (1995) J. Biol. Chem. 270, 29945-52.
6. Strauss, J. K., Roberts, C., Nelson, M. G., Switzer, C. and Maher, L.
J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520.
Ada Yonath
Department of Structural Biology,
Weizmann Inst.,
Rehovot and Max-Planck Unit for Ribosome Structure, Hamburg
*For author correspondence. Phone: 972-8-9343028, Fax: 972-8-9344154; E-mail:csyonath@WEIZMANN.WEIZMANN.AC.IL
The translation of the genetic code into polypeptide chains is performed on giant riboprotein organelles called ribosomes. In prokaryotes about 1/3 of its mass is comprised of some 58-73 different proteins, depending on the organizm. The remaining 2/3 is made of three chains of rRNA, with a total of about 4500 nucleotides. These are arranged in two independent subunits of unequal size (0.9 and 1.45 mega dalton, respectively) which associate upon the initiation of protein biosynthesis. The small subunit provides the sites for the decoding of the genetic information and for the control of the fidelity of the process. The large subunit catalyzes the formation of the peptide bond and provides the progression path of the nascent proteins.
Crystals, diffracting to 3 A resolution were grown from small and large ribosomal subunits of Thermus thermophilus and Haloarcula marismortui, respectively. Data collected to 3.8-7.5 A at cryo temperature with bright synchrotron beam led to MIRAS-phased electron density maps showing shapes similar to the corresponding EM reconstructions and revealing recognizable key internal features (e.g. the large subunit tunnel) and contain (a) dense elongate continuous regions spanning the particles at various directions and showing typical tertiary and secondary-structure features of RNA: single strands and well separated double helices; and (b) globular and ellipsoidal regions of lower density, appropriate for the accommodation of ribosomal proteins, some of which show the main-chain folds observed in crystals of isolated ribosomal proteins. Tracing the RNA chains, assignment of the protein domains, and analyzing their architectural relationships, are underway.
For unbiased map interpretation, heavy atom markers are being attached directly to the particles or through cDNA and/or antibiotics. So far the location of a selected cysteine and several surface RNA strands were elucidated.
Molecular replacement studies using cryo-EM reconstructions confirmed the MIR crystal packing motifs and the major heavy atom sites, provided a tool for assessing non-isomorphism, the less tolerable property of H50S and indicated close to active conformation of crystallized T30S. Consequently crystals of increased quality is obtained by reactivation within the crystals or by crystallizing particles trapped at their activated state.
To facilitate higher resolution phasing, extensive selenation,
suitable for MAD phasing is being introduced by recombinant methods. As
totally assembled ribosomal particles did not yield well diffracting crystals,
we focus on proteins that can be quantitatively and reversibly detached,
by chemical means, mutations or gene knockouts. The same proteins are also
being subjected to pre-crystallization heavy atom binding.
Günther Burckhardt1, Ingo Förtsch1, Hannelore Simon1, Eckhard
Birch-Hirschfeld2, Leonhard Kittler3, J.William Lown4 and Christoph
Zimmer1
1Institut für Molekularbiologie der Friedrich-Schiller-Universität
(FSU) Jena
2Institut für Virologie des Klinikums der
FSU Jena,
Winzerlaer Str. 10, D-07745 Jena, Germany
3Institut für Molekulare Biotechnologie
e.V.,
Beutenbergstraße 11, D-07745 Jena, Germany
4Department of Chemistry,
University of Alberta,
Edmonton, Canada T6G2G2
The sequence recognition in the DNA minor groove has been improved by the finding that two distamycin A molecules could bind by a side-by-side motif to opposite strands in this groove (1). It was further demonstrated that polyamide lexitropsins containing the imidazole GC-reading element can differentiate between GC and CG base pairs by this side-by-side drug binding mode (2). As a possible discriminating element for AT versus TA a bulky thiazole(Th)ring in combination with pyrrole(Py)rings has been proposed (2). By using circular dichroism we have made an attempt to determine whether it is possible to get insights into the DNA binding specificity of a cross-linked lexitropsin composed of two (thiazole-imidazole-pyrrole) strands, and which could fit in the minor groove (3). The binding of this polyamide lexitropsin containing a heptanediyl linker (C7 dimer) with monocationic strands was compared with that of the monomer. CD binding measurements with 28 oligodeoxyribonucleotide duplexes of various sequences reveal several significant results. Using the induced CD maxima as drug binding signals some distinct differences appear in the titration profiles for AT- and GC-rich sequences with the C7 dimer at 200 mM NaCl. However, at 2M NaCl the CD binding behavior of the dimer is completely changed for different sequences (for a shortened notion we refer to one strand of the central segment of the dodecamer duplex as the expected binding sequence e.g. for d(CGCAAATTTGCG): - AAATTT-). The largest binding effects of the dimer in 2M NaCl occur to the sequences
- AACGTT- ; -ATCGTA- ;
- AAAGAA- ; -AAAGTT-
but almost no binding is observed for -AAATTT- and -AGCGCT-.
Two aspects can be visualized from the studies: (i) the sequence selectivity is highly increased for the polyamide dimer, which is not the case for the monomer and (ii) modification of the leading group in the dimer structure can lower (or even abolish) its sequence binding ability. Footprinting and melting temperature data agree with the CD findings. Possible binding schemes of the dimer-DNA oligomer complex will be proposed. In vitro studies with DNA gyrase show that the dimer has a significantly higher inhibitory potency on the enzymatic activity compared to the effect of the monomer.
References and Footnotes
1. Pelton, J.G. and Wemmer, D.E., (1989) Proc. Natl.
Acad. Sci. USA 86, 5723-5727.
2. Kopka, M.L., Han G.W., Goodsell, D.S., Chin, T.K., Walker, W.L., Lown,
J.W. and Dickerson, R.E., Proceeding 10th Conversation, State Univ.
NY, Albany, NY1997 (Eds. R.H.Sarma, M.H. Sarma) Adenine Press 1998, 177-191.
3. Chen, Y.-H., Yang, Y. and Lown, J.W. (1996) J. Biomolec. Struct. Dyn.14,
341-355.
Helen G. Hansma
Department of Physics,
University of California,
Santa Barbara, CA 93106
This talk will cover recent developments in our lab on atomic force microscopy (AFM) (1,2) of DNA including:
[1] The analysis of DNA condensed for receptor mediated gene therapy
with covalent complexes of polylysine with asialoorosomucoid (3). The DNA
condenses in stages, depending on the type and quantity of polycation, into
compact toroids. These toroids appear to contain much less DNA per unit
volume than the classical toroids described by Bloomfield and others (4).
[2] The direct visualization of DNA transcription with E. coli RNA polymerase
(5,6).
[3] Evidence that the CBF3-CEN DNA complex of the yeast kinetochore is involved
in holding chromosome pairs together during cell division (7).
References and Footnotes
1. Hansma, H. G., and Pietrasanta, L., Atomic force microscopy
and other scanning probe microscopies, Current Opinion in Chemical Biology,
2, 579 (1998).
2. Colton, R. J., Baselt, D. R., Dufrene, Y. F., Green, J. B. D., and Lee,
G. U., Scanning probe microscopy, Current Opinion in Chemical Biology,
1, 370 (1997).
3. Hansma, H. G., Golan, R., Hsieh, W., Lollo, C. P., Mullen-Ley, P., and
Kwoh, D., DNA condensation for gene therapy as monitored by atomic force
microscopy, Nucleic Acids Res., 26, 2481 (1998).
4. Arscott, P. G., Li, A. Z., and Bloomfield, V. A., Condensation of DNA
by trivalent cations. 1. Effects of DNA length and topology on the size
and shape of condensed particles, Biopolymers, 30, 619 (1990).
5. Kasas, S., Thomson, N. H., Smith, B. L., Hansma, H. G., Zhu, X., Guthold,
M., Bustamante, C., Kool, E. T., Kashlev, M., and Hansma, P. K., E. coli
RNA polymerase activity observed using atomic force microscopy, Biochemistry,
36, 461 (1997).
6. Stokstad, E., DNA on the big screen [news], Science, 275, 1882
(1997).
7. Pietrasanta, L. I., Thrower, D., Hsieh, W., Rao, S., Stemmann, O., Lechner,
J., Carbon, J., and Hansma, H. G., Probing the Sacchromyces cervisiae CBF3-CEN
DNA kinetochore complex using atomic force microscopy, Proc. Natl. Acad.
Sci. (USA), in press (1999).
Supported by NSF MCB 9604566.
Udo Heinemann1*, Uwe Mueller1, Hermann Heumann2 and Mathias
Sprinzl3
1Forschungsgruppe Kristallographie,
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str.
10,
D-13125 Berlin, Germany,
and Institut für Kristallographie,
Freie Universität Berlin, Takustr. 6,
D-14195 Berlin, Germany
2Max-Planck-Institut für Biochemie, Am
Klopferspitz 18A,
D-82152 Martinsried, Germany;
3Laboratorium für Biochemie,
Universität Bayreuth,
Universitätsstr. 30,
D-95440 Bayreuth, Germany
*Author to whom correspondence should be addressed. Phone: +49 30 9406 3420;
Fax: +49 30 9406 2548; E-mail: heinemann@mdc-berlin.de
The determinants for specific protein binding to RNA are studied at high resolution by crystal structure analysis of synthetic oligoribonucleotide helices. In two examples presented here, the RNA shows standard A-form geometry in the crystal, whereas it is reported to have a flexible, or non-standard conformation in solution based on NMR studies.
The crystal structure of an eight-base-pair chimeric duplex representing the 3 '-DNA-RNA-5 ' junction formed during initiation of minus-strand DNA synthesis by HIV-1 reverse transcript-ase was determined at 1.9Å resolution (1). A base-pair step with distinct stacking geometry and variable backbone conformation is found next to the main site of endonucleolytic cleavage by HIV-RT associated RNase H. It is suggested that this base-pair step serves as a recognition element for the ribonuclease.
RNA duplexes representing the acceptor stem of Escherichia coli tRNAAla and its G3·U70> G3-C70 mutant were investigated in order to assess the structural role of the unique G·U base pair which has been shown to be crucial for specific binding by alanyl-tRNA synthetase. The crystal structures were solved using disordered or twinned crystals at 1.16 and 1.4Å resolution (2). In the wild-type double helix, the crystal lattice is stabilized by a novel Sr2+ binding site. Significant structural differences between the two duplexes are seen only at the site of the G3·U70 wobble base pair which displays a highly ordered solvent structure in both grooves which we suggest to be of functional relevance.
Supported by the Deutsche Forschungsgemeinschaft through He 1318/12 and the Fonds der Chemischen Industrie.
References and Footnotes
1. Mueller, U., Maier, G., Mochi Onori, A., Cellai, L.,
Heumann, H. & Heinemann, U., Biochemistry 37, 12005-12011, 1998.
2. Mueller, U., Sch¸bel, H., Sprinzl, M. & Heinemann, U. (1999)
RNA, in press.
Chang-Beom Cho1, Sung Wook Han2 and Seog K. Kim1
1Department of Chemistry,
Yeungnam University,
Kyoungsan City,
Kyoung-buk, Republic of Korea
2Department of Environmental Engineering,
Kyungwoon University,
Kumi City,
Kyoung-buk, Republic of Korea
There have been debate on the binding mode of [Ru(phenanthroline)2L]2+ to DNA. It was proposed by Kim
et al. that the metal complex intercalates from the minor groove of the
double stranded DNA (Biochemistry, 36, 214-223, 1997). This proposal
based on the observation that the binding mode of the metal complex to double
stranded DNA was identical with that to triple helical DNA to which the
third strand blocks the major groove. However, Barton et al. argued that
the metal complex intercalates from the major groove (Inorg. Chem., 37,
29-34, 1998). Supporting evidence for this assumption is that the binding
of the major groove binder Delta-alpha-[Rh[(R,R)-Me2Trien]Phi]3+ results in decreases in the emission yield of the [Ru(phenanthroline)2L]2+-DNA complex, while the minor
groove binding drug distamycin has no effect. In contrast, we observed that
the fluorescence intensity of the complex remained to be unchanged by addition
of 4,6-diamidino-2-phenylindole. Furthermore, addition of the ruthenium
complex results in the releasing of this drug. This result disagrees with
latter observations and supports the direct interaction of ruthenium complex
with minor groove of DNA.
Young Ae Lee1, Sung Wook Han2 and Seog K. Kim1
1Department of Chemistry,
Yeungnam University,
Kyoungsan City,
Kyoung-buk, Republic of Korea
2Department of Environmental Engineering,
Kyungwoon University,
Kumi City,
Kyoung-buk, Republic of Korea
We studied Mg2+ and ATP dependent thermal stability
of Rad51 protein in the presence and absence of single stranded DNA using
CD spectroscopy. Mg2+ ion directly binds to Rad51 protein
and activated it for the binding of single stranded DNA. The activated Rad51
protein shows two clear steps in the melting profile: the first step in
20-40°C and the second at 80°C. Although ATP alone does not affect
the melting profile of the Rad51 protein, it lowered the second melting
temperature when it is co-exist with Mg2+, indicating
Mg2+ mediated ATP binding to Rad51 protein. Single
stranded DNA binds to Rad51 in the presence of Mg2+
and ATP and binding of ssDNA to the protein inhibits the denaturation step
as well as the first transition. The temperature-dependent Rad51 protein
conformation will be discussed.
Mihai Ciubotaru1, Frank V. Bright2, Christine Ingersoll2 and
Gerald B. Koudelka1
1Department of Biological Sciences,
2Department of Chemistry,
SUNY at Buffalo,
Buffalo, NY 14260-1300
Although 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. To begin to resolve these apparently paradoxical observations, we have used steady-state fluorescence and fluorescence anisotropy measurements to study the structure 434 repressor in the absence and presence of various DNA binding sites. The results of these investigations suggest that both specific and non-specific DNA induce conformational changes in repressor that leads to formation of repressor dimers. Our data also show that the repressor conformational changes induced by specific and nonspecific DNA occur at DNA concentrations much lower than those needed to bind repressor. This finding suggests that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. This suggestion implies that even brief collisions between 434 repressor and DNA induce these conformational changes. Although we have not yet determined the lifetime of the DNA-induced conformations, time resolved DNA anisotropy measurements show that in the case of the nonspecific DNA, the protein-DNA contact that affects repressor structure has a lifetime of <3 nsec. Hence our data indicate that DNA acts in a "catalytic" fashion to induce a steady state amount of an alternative repressor conformation.
In effort to discern the functions of the dimeric repressor forms induced by nonspecific DNA, specific DNA or increased repressor concentrations, we measured the relative affinities of these protein species for a specific binding site. Our data show that the repressor dimer species formed in the presence of specific or non-specific DNA has an enhanced affinity for specific DNA, and that the dimer species formed at higher repressor concentration has a lower affinity for specific DNA. We find that the dimeric species induced by high repressor concentrations is not formed in vivo. We considered these observations in an attempt to discern a possible pathway for the assembly of repressor-DNA complexes. We suggest that the repressor conformer induced by non-specific-DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein an additional conformational change, that allows it to "lock-on" to its specific site. We will present additional evidence supporting the existence of this pathway and that elucidates the functional role of each repressor conformer.
Vitaly V. Kuryavyi and Dinshaw J. Patel
Cellular Biochemistry and Biophysics Program,
Memorial Sloan-Kettering Cancer Center,
New York, NY, 10021
Usually in a DNA structure the number of bases interacting in-plane is equivalent to the number of strands crossing this plane (i.e.: 2 bases = 2 strands in a duplex, 3 bases = 3 strands in a triplex, 4 bases = 4 strands in a quadruplex). Triad DNA escapes this rule (1). For 3 bases in its unit, called "triad", 2 strands are sufficient. This duplex features "triangularity" (2) what makes it an elegant yet hypothetical intermediate in triplet repeat expansion disorders.
The recent experimental verification of several triad configurations: (3,4,5) both in DNA and RNA molecules has evoked a number of approaches to design sequences toward discovery of other individual base triads and possible interactions they are able to engage into.
This paper addresses a generalized issue. Is triad DNA an exceptional structure featuring base and strand non-equivalency or just a first member in the family? The modeling work into this problem revealed a number of stereochemical possibilities where base:strand ratio is similar to the triad (>1) or inverted (<1). The fractionality of this value is characteristic for this class of DNA structures.
Different models composed of isomorphic units C-T-G and C-C-G, which are triplets with T and C interacting at the minor groove side of the GC base pair will be presented:
a) >1 group
Figure 1: The symmetric fold gives rise to a structure called "Tirad DNA" in which the backbone progresses up-down-up with interdigitation and triplet repeat (GCC)n, or (CTG)n.
Figure 2: An asymmetric fold causes a structure whose 6 bases are distributed between two strands in the ratio 4:2. The symmetric connection of these two units results in a structure with hexanucleotide repeat (CGCCCG)n.
b) <1 group.
Figure 3: A structure composed of two hairpin-like antiparallel interacting folds with three bases keeping them together through the triple interaction.
Association with the Institute of Theoretical and Experimental Biophysics RAN 142292 Pushchino Moscow Region is acknowledged.
References and Footnotes
1. Kuryavyi, V.V. & Jovin, T.M. Triad-DNA: a model
for trinucleotide repeats. Nature Genetics 9, 339-341 (1995).
2. Kuryavyi, V.V. & Jovin, T.M. Triangular complementarity of the Triad-DNA
duplex. Proceedings of the Ninth Conversation, Eds. R.H.Sarma and
M.H.Sarma, Adenine Press p. 91-103 (1996).
3. Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Szewczak,
A.A., Kundrot, C.E., Cech, T.R. & Doudna, J.A. RNA tertiary structure
mediation by adenosine platforms. Science 273, p. 1696-1699 (1996).
4. Kettani, A., Bouaziz, S., Wang, W., Jones, R.A. & Patel, D.J. Bombyx
mori single repeat telomeric DNA sequence forms a G-quadruplex capped by
base triads. Nature Structural Biology, 5, p. 382-389 (1997).
5. Zimmermann, G.R., Jenison, R.D., Wick, C.L. Simorre, J.P, & Pardi,
A. Interlocking structural motifs mediate molecular discrimination by a
theophylline-binding RNA. Nature Structural Biology, 4, p. 644-647
(1997).
J.Y. Ostashevsky, B.I. Reichman and C.S. Lange*
SUNY - Downstate Medical Center,
Box 1212, Brooklyn, NY 11203
*Author to whom correspondence should be addressed. Phone: (718) 270-1050;
Fax: (718) 270-1608; E-mail: Lange_c@hscbklyn.edu
In a recent model of G1 chromosome structure (Ostashevsky, Mol. Biol.
Cell 9:3031, 1998) the 30 nm chromatin fiber of each chromosome forms
a string of loop clusters (micelles). Here we show for plateau phase V79
Chinese hamster lung fibroblasts (unirradiated and X-irradiated with doses
up to 40 Gy) that viscoelastometry (VE) can yield two parameters of the
model - the number of loops per micelle (f ) and the loop's size
(Mf). Despite extensive cell lysis (1 h in 2
M NaCl + proteinase K + sarkosyl at 50°C), the ratio of A/G (Area
under the VE recoil curve/Total recoil amplitude), an analog of the relaxation
time constant, is two orders of magnitude smaller (~11 ksec) than that expected
for a linear random coil configuration (~1800 ksec). As dose increases,
A/G decreases smoothly to 3 ksec. Assuming that intermicelle DNA double-strand
breaks (DSBs) shorten molecules while DSBs in the loops do not affect VE
parameters, the best fit of A/G data for 2 M NaCl solution yields
f = 16-18 loops per micelle, which is similar to the estimates (f
~ 10-20) made by applying the model to other data, particularly those from
Yokota et al. (J. Cell Biol. 130:1239, 1995) obtained by FISH technique
for human chromosome # 4. For lysis in 0.2 M NaCl, where DNA-bound proteins
are not completely removed, A/G increases from the initial 3 ksec
for unirradiated cells to a peak of 5 ksec at 4 Gy and then decreases in
parallel to the data for lysis in 2 M NaCl. The initial increase in A/G
may be due to enhanced protein removal by DNA single-strand breaks. These
data yield a loop size of ~1.3 Mbp, which is close to literature values
estimated by other means.
Elena A. Lesnik*, Nathan B. Sioufi and Susan M. Freier
ISIS Pharmaceuticals,
2292 Faraday Avenue,
Carlsbad, CA 92008
*Author to whom correspondence should be addressed. Phone: (760) 603-2377;
Fax: (760) 931-0209; E-mail: elesnik@isisph.com
Antisense oligodeoxynucleotides (ODNs) have been shown to inhibit gene expression for a number of cellular targets (1). The first generation of ODNs is phosphorothioate oligonucleotides which are more stable in vivo than their phosphodiester counterparts and demonstrates pharmacological activity in animal models and human beings (2). An understanding of the forces governing the ODN binding to serum proteins and especially to human serum albumin (HSA) which is believed to play a key role in the transport of the ODNs to the targets is an important for further development of RNA related medicines and molecular biology tools.
There are fragmentary contradictory data on ODN binding to HSA (3-5) obtained by different methods at different HSA concentrations. Albumins have been shown to be inclined to self-association resulting in the concentration dependence of dissociation constants (Kd) for some ligands (6,7). We made an attempt to elucidate the nature of the ODN-HSA binding, the number of binding sites and calculate Kd for ODN binding over a wide range of defatted HSA concentrations [15-600 uM] using a microfiltration method. Results obtained from salt dependence studies suggested that non-ionic interactions play a major role in phosphorothioate oligonucleotide nonspecific binding to albumin. Two binding sites with Kd values 3-6 uM and 80-120 uM respectively were found. In contrast to small molecule ligands, both binding sites bind oligonucleotides with low capacity. Possible explanations will be discussed.
References and Footnotes
1. Crooke, S.T., Ann. Rev. Pharmacy. Toxic. 32,
329-376, 1992.
2 Stepkowski S. M. et al., Transplantation, 66, 699-707, 1998
3. Srinivasan S. K. et al., Antisense Res. and Development, 5, 131-139,
1995.
4. Greig M. J. et al., JACS, 117, 10765-10766, 1995.
5. Crooke S. T. et al., J. Pharmac. and Experm.. Therapeutics, 277,
923-937, 1996.
6. Bowmer, C. J. et al., Biochem. Biophys. Acta, 624, 260-270, 1980.
7. Brors O. et al., Pharmacology & Toxicology, 72, 310-313, 1993.
Patrick Furrer, Rob Manning and John Maddocks*
Department of Mathematics,
EPFL (Swiss Federal Institute of Technology),
1015 Lausanne, Switzerland
*Author to whom orrespondence should be addressed. Phone: +4121-693262,
E-mail: maddocks@dma.epfl.ch
How many stable equilibria (either cyclized or nicked) will a DNA loop have? With an intrinsically straight and transversely isotropic rod model of DNA, the answer is "one". However, when intrinsic curvature is taken into account, the answer can change, as this family of equilibria may in principle yield any number of stable equilibria. This phenomenon was reported in a recent Monte Carlo study (V. Katritch and A. Vologodskii, Biophysical Journal 72 , 1070-1079, 1997), but without fundamental explanation of its cause.
This multiplicity can be understood by analysis within the rod model. A perturbation study of the rod equations yields simple multiplicity conditions, describing which infinitesimal intrinsic curvatures can yield multiple stable equilibria. In a study of several thousand random 200 or 900 base-pair DNA sequences, we have found that these multiplicity conditions remain good predictors of multiple equilibria even for these quite high intrinsic curvatures.
We also analyze the connections between continuum equilibria and distributions of shapes generated by Monte Carlo simulations. We find surprisingly good correlations between Monte Carlo Lk peaks and Lk values of continuum equilibria, andeven between Monte Carlo peak intensities and continuum energies, despite the fact that Monte Carlo visits many more shapes than just the equilibria.
J. W. Bizzaro and Kenneth A. Marx
The BIC Group
28 Pope Street
Hudson, MA 01749
www.bicgroup.org
Loci is a computer software framework for linking bioinformatics and computational biology programs of very disparate types to a collection of graphical and analytical tools. The software is targeted at numerous diverse groups, covering practically anyone with an interest in the molecular level of biology. One of the major features of Loci, is the ability to attach a graphical user interface (GUI) to nearly any command-line program or database. These can therefore be distributed throughout the Internet or intranet and accessible to anyone using Loci, providing the researcher with a suite of tools unlimited in expansibility. Users of the software can also collaborate with each other anywhere across the Internet or intranet. These features and many others were made possible by implementing software technologies that are rather new to biocomputing, including work flow and distribution, which can both be choreographed and monitored using Loci's powerful GUI. But perhaps the most remarkable feature of Loci, is that all of this power and flexibility, unseen in even the most expensive commercial packages, costs nothing. More information can be found at www.bicgroup.org.
Tiean Zhou1, Kenneth A. Marx1*, Michael Warren2 and Susan
J. Braunhut2
Center for Intelligent Biomaterials,
1Department of Chemistry,
2Department of Biological Sciences
University of Massachusetts,
Lowell, MA 01854
*Author to whom correspondence should be addressed. Phone (978) 934-3658
; Fax (978) 934-3013; E-mail: @uml.edu
Endothelial cells(ECs), in vivo, form a continuous non-dividing monolayer lining the interior surface of blood vessels. In large vessels, these cells can experience significant pulsatile blood flow pressures (110 mm Hg/s). Therefore, stable EC attachment to its extracellular matrix represents an important biological state. We have previously demonstrated that ECs can attach to optically transparent metallic electrode substrates and that, subsequently, they can be growth stimulated at -0.3 V and growth inhibited at +0.2 V (1). In order to focus our study of the EC adhesion process, we have applied the Quartz Crystal Microbalance(QCM) technique (2,3) to the real time continuous monitoring of ECs adhering to and spreading on the QCM gold surface. We have investigated a number of parameters associated with this phenomenon including: variability due to different QCM crystals and the effects of hydrophilic treatment of the gold surface; determining different O-ring toxicities toward ECs; frequency oscillation in the measurements and its elimination. The dependence of the measured frequency (f) and motional resistance (R) on the number of firmly attached ECs was established. EC numbers were measured by electronic counting of cells requiring trypsinization for their removal from the surface, as opposed to assuming that all added ECs were attached at 24 hrs. We demonstrated steady state Deltaf and DeltaR shift values for the stably attached ECs at 24 hr post-addition (4). Finally, through a light microscope simulation experiment, we showed that the different Deltaf and DeltaR regions of the QCM response curve correspond to the phenomena of EC adhesion, spreading and cellular mass distribution changes. Supported by UML Seed Funds and NIH R21 GM58583.
References and Footnotes
1. Zhou, T., Braunhut, S.J., Medeiros, D. and Marx, K.A.,
in Materials Science of the Cell, Proceedings Materials Research Society,
489, 211-216 (1998).
2. Ward, M.D., Principles and Applications of the Electrochemical Quartz
Crystal Microbalance, in Physical Electrochemistry, Principles, Methods
and Applications, Marcel Dekker, 293-338 (1995).
3. Marx, K.A., Zhou, T. and Sarma, R., Biotechnology Progress, 15,
in press (1999).
4. Zhou, T., Braunhut, S.J., Medeiros, D. and Marx, K.A., in Tissue Engineering,
Proceedings Materials Research Society, in press (1999).