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* Department of Chemistry and Biochemistry, Center for Nano and Molecular Science and Technology, University of Texas, Austin, Texas 78712; Department of Applied Physics, Osaka University, Suita, Osaka 565 0871, Japan; and Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
Correspondence: Address reprint requests to Paul F. Barbara, E-mail: p.barbara{at}mail.utexas.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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; South et al., 1990; Summers et al., 1992) nucleic acid chaperone protein, which facilitates reverse transcription of the HIV-1 RNA genome by catalyzing the rearrangement of nucleic acid structures (Darlix et al., 1995; Herchslag 1995; Lorsch 2002; Rein et al., 1998; Tsuchihashi and Brown 1994). To achieve such rearrangements, certain basepair interactions must be broken, while other complementary basepair interactions must be formed, resulting in a thermodynamically more stable conformation.
Various steps in reverse transcription are catalyzed by NC. One such step is minus-strand strong-stop DNA ((–) SSDNA) strand transfer to the 3' end of the RNA genome. This reaction is mediated by basepairing of the complementary repeat regions in (–) SSDNA and viral RNA. This process involves annealing of the transactivation response (TAR) RNA present in the viral RNA, to the complementary DNA sequence (TAR DNA) in (–) SSDNA. Both TAR RNA and TAR DNA sequences are predicted to fold into stable hairpin structures (Coffin et al., 1997; Guo et al., 1997; Kim et al., 1997; You and McHenry, 1994). These hairpin structures prevent the intermolecular annealing reaction resulting in the formation of a 98-nucleotide (nt) basepaired binary complex. NC has been shown to stimulate minus-strand transfer by increasing the rate of intermolecular annealing and by blocking a competing intramolecular self-priming reaction (Driscoll and Hughes, 2000; Guo et al., 1997; Lapadat-Tapolsky et al., 1997; You and McHenry, 1994), which occurs due to the presence of the TAR DNA hairpin at the 3' end of the (–) SSDNA (Coffin et al., 1997; Guo et al., 1997; Kim et al., 1997; You and McHenry, 1994).
The effect of NC binding on the conformations of TAR DNA hairpins has previously been investigated by ensemble fluorescence resonance energy transfer (FRET) measurements and other techniques (Azoulay et al., 2003; Beltz et al., 2003; Bernacchi et al., 2002; Hong et al., 2003). These data demonstrate that bound NC shifts the TAR DNA hairpin equilibrium toward "open" conformations. However, the averaging inherent in ensemble FRET measurements makes it difficult to identify specific conformations (Azoulay et al., 2003; Beltz et al., 2003; Bernacchi et al., 2002; Hong et al., 2003). To obtain more detailed information on the conformations of TAR DNA/NC we undertook the first time-resolved single molecule fluorescence resonance energy transfer (SM-FRET) measurements on TAR DNA hairpins and hairpin mutants in the presence and absence of NC protein. Our single molecule results clearly demonstrate that bound NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully "closed" conformation to a specific "partially open" conformation with the two terminal stems "open" or unwound and the other stems closed. In addition, the data show that the two terminal stems in the TAR DNA/NC complex undergo a rapid opening/closing process. The observed partially open TAR DNA/NC conformation seems ideally suited for the promotion of NC catalyzed DNA/RNA annealing, because the open terminal stems possess 21 unpaired bases accessible for DNA/RNA annealing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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). The dye-labeled DNA hairpins (64 mer, 67 mer, 68 mer, 70 mer, and 71 mer) were purchased from TriLink Biotechnologies (San Diego, CA), and their predicted secondary structures are shown in Fig. 1. Cy3 and Cy5 were chosen as the FRET donor and acceptor dyes, respectively. Cy3-amidite was directly coupled to the 5' end of the DNA. Cy5-succinimidyl ester was postsynthetically coupled to an amino linker at the 3' end of the DNA. To prevent the undesirable G residue quenching effects (Kurata et al., 2001; Torimura et al., 2001), a 5'-T and 3'-TTTT overhang were added to the core 59-mer TAR DNA sequence. The 3' overhang also prevents formation of a dark nonfluorescent state that can form due to close proximity of the two dyes (see also Fig. S1 and the accompanying discussion in the Supplementary Material) (Bernacchi et al., 2002). To study fluorescence intensity time trajectories over long time periods, DNA hairpins were immobilized using the biotin-streptavidin interaction (Grunwell et al., 2001; Ha et al., 1999; Zhuang et al., 2000). A biotin was internally attached via a dT-biotin phosphoramidite reagent. The oligonucleotides were purified by the supplier by polyacrylamide gel electrophoresis and reversed-phase HPLC. Before hairpin immobilization on the coverslips (see below), the oligonucleotide solution was diluted to a final concentration of 25–50 pM in HEPES buffer (25 mM HEPES, pH 7.3, 40 mM NaCl) containing 10 mM MgCl2 and reannealed by incubation for 2.5 min at 80°C, 2.5 min at 60°C, and 5 min at 0°C.
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10 µL. Inlet and outlet ports (Nanoport, Upchurch Scientific, Oak Harbor, WA) were glued on top of the chambers. The chambers were incubated sequentially for 10 min with solutions of biotinylated bovine serum albumin (BSA) (Pierce Biotechnology, Rockport, IL; 2 mg/mL in distilled deionized water) and streptavidin (Molecular Probes, Eugene, OR; 0.2 mg/mL in HEPES buffer). The chambers were rinsed with distilled deionized water (50 µL) after each incubation step. The doubly labeled oligomer solution (25–50 pM) was incubated subsequently for 20 min. The chambers were rinsed and Teflon tubing was adapted to the chamber ports. Two syringe pumps delivered solutions at a rate of 2 µl/min. A 5-min equilibration period at a flow rate of 10 µl/min was elapsed before measurements were taken under a new set of conditions (e.g., addition of NC). All solutions consisted of HEPES buffer containing 0.2 mM MgCl2 and an oxygen scavenger system (2-mercaptoethanol 1% v/v (Sigma-Aldrich, St. Louis, MO), ß-D(+)glucose 3% w/v (Sigma-Aldrich), glucose oxidase 0.1 mg/mL (Roche Applied Science, Hague Road, IN), and catalase 0.02 mg/mL (Roche Applied Science)) (Ha, 2001; Harada et al., 1990). Single molecule spectroscopy on the dye-labeled hairpins with polarized excitation and modulation of the direction of polarized light demonstrated that the dyes were freely rotating (data not shown). This is consistent with successful immobilization of the hairpins without undesirable interactions of the hairpin-dye structures with the surface of the coverslip.
Experimental setup
The experimental setup has been described previously (English et al., 2000; Yip et al., 1998). A closed-loop sample scanning stage (NPS-XY-100A, Queensgate, Torquay, Devon, UK) was used for imaging and sample positioning. Continuous wave excitation (514 nm, 5–10 µW/µm2) from an argon ion laser (model Reliant 150m, Laser Physics, West Jordan, UT) was introduced via an optical fiber and was directed by a dichroic beamsplitter (530 DCLP, Chroma, Rockingham, VT) to the sample via a high numerical aperture (N.A.) oil immersion microscope objective (Zeiss Fluar, 100x, N.A. 1.3) (Carl Zeiss, Oberkochen, Germany). Fluorescence from the sample passed through the dichroic beamsplitter and a holographic Raman notch filter (Kaiser Optical Systems, Ann Arbor, MI) and was then directed to the detectors by means of an internal mirror. Imaging of the sample and measurement of single molecule fluorescence intensity time trajectories were conducted using two avalanche photodiode (APD) detectors (Perkin Elmer Optoelectronics SPCM-AQR-15, Vaudreuil, Quebec, Canada). The collected fluorescence was separated into donor and acceptor channels by a dichroic beam splitter (Chroma 630 DCXR). The TTL output signal from the APDs was distributed by a 1:4 fanout TTL driver (Pulse Research Lab, Torrance, CA) into an ALV 5000/E (ALV-Laser, Langen, Hessen, Germany) correlation board and a counter board. This configuration allowed us to simultaneously obtain intensity time trajectories with 1-ms time resolution and donor-acceptor intensity cross correlation with 0.5-µs time resolution for each single molecule.
Data analysis
Single hairpin fluorescence intensity time trajectories were recorded using separate donor and acceptor channels, each with an APD detector. The signals Si(t) were corrected for background emission and donor/acceptor cross talk due to overlapping emission using Eq. 1:
 | (1) |
). The simultaneous detection of both donor ID(t) and acceptor IA(t) fluorescence intensity time trajectories can be used to calculate the time trajectory of the FRET efficiency, EFRET(t), as shown in Eq. 2, where 
A and D are the emission quantum yields of acceptor and donor dyes, respectively, and 
A and D are the acceptor and donor detector efficiencies, respectively. Single molecule EFRET(t) trajectories reflect the time trajectory of the end-to-end distance R(t) in the donor-acceptor pair as observed in Eq. 3 (Lakowicz, 1999):
 | (2) |
 | (3) |
).
The correction factor for emission and detection efficiencies (A x A / D x D) is 1 under our experimental conditions, thus the apparent FRET efficiency Eapp(t), given by the ratio of acceptor to the sum of acceptor plus donor intensities is equal to EFRET(t). The correction factor was determined by comparing the emission from acceptor dye under 100% energy transfer efficiency and the emission from the donor-only subpopulation of molecules.
Blinking events (acceptor reversible photobleaching) were removed before data analysis. A threshold criterion was applied to the counts on the acceptor channel. This criterion removes experimental points where the acceptor intensity is due to background and/or cross talk from the donor channel. The threshold criterion equals 2x the combined uncertainty on the acceptor channel (SA) resulting from the background (SBA) and the cross talk (SCA), as shown in Eq. 4.
 | (4) |
Assuming that the only source of uncertainties is photon shot noise in the background and the cross talk, Eq. 4 rearranges to:
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BA
is the average background intensity in the acceptor channel and CA is the average cross talk from donor into acceptor channel before acceptor photobleaching. (Note that for simplicity we have approximated CA as a constant value; CA is, however, dependent on the donor intensity, and therefore on hairpin end-to-end distance fluctuations).
Time intervals where acceptor counts were lower to or equal to the threshold value (2 x (BA + CA)(1/2)) were removed from the intensity time trajectories (see also Fig. S2 in Supplementary Material).
Single molecule cross correlation of ID(t) versus IA(t) were calculated with Eq. 5.
 | (5) |
Each single molecule cross correlation curve was normalized to Eapp autocorrelation under the assumption that 
that is: 
Under this assumption the Eapp autocorrelation equals the IA autocorrelation (Eq.6).
 | (6) |
Replacing 
ID(t) by –IA(t) in Eq. 5 we get:
 | (7) |
Multiplying Eq. 7 by (–ID/IA) the cross correlation is normalized to the Eapp autocorrelation; see Eq. 8:
 | (8) |
We averaged the single molecule Eapp autocorrelation curves obtained under a set of experimental conditions (with or without NC).
Donor-acceptor cross correlations were also obtained with an ALV 5000/E correlation board with 0.5-µs time resolution. Data were acquired for a total of 4 s. The data from any single molecule that photobleached before finishing acquisition were discarded.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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; Williams et al., 2001). Maximum annealing rates between TAR DNA and TAR RNA hairpins occur for [NC] 
500 nM (Guo et al., 2002). Our experiments were performed with saturating NC concentrations close to this range.
Any single molecule is representative of the ensemble under our experimental conditions, i.e., the system is ergodic. Individual single molecule trajectories and distributions of Eapp(t) are indistinguishable from each other when recorded under the same experimental conditions. The mean Eapp values measured for individual single molecules (Eappmol) are also very similar to each other (Fig. 2, left column; see also Fig. S3 in Supplementary Material).
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Eappmol 0.77, 
Eapp = 0.07 was measured for TAR DNA single molecules with NC flowed at a rate of 2 µl/min (Fig. 2 A). The same values were obtained for mutant forms of TAR DNA hairpins for which the internal bulge L4 (see Fig. 1 B) and both internal bulges L4 and L3 (see Fig. 1 C) were deleted (see Fig. 2 B). Inspection of the single molecule trajectories of Eapp(t) reveals that the hairpins sporadically closed to the conformation with Eapp 1 (see right columns in Fig. 2, A and B). The closing events are also evident from the unsymmetrical distribution of Eapp(t) (see Fig. 2, A and B). From the distribution of Eapp(t), as much as 10% of the events can be assigned to a closed hairpin conformation.
NC induces larger donor-acceptor dye separations in a structure with two internal bulges compared to one internal bulge. Thus the distribution of Eapp(t) for a third TAR DNA mutant form conserving only the terminal internal loop (-L4-L3-L2, Fig. 1 D) has a mean value Eappmol = 0.90, Eapp = 0.05 with 445 nM NC (see Fig. 2 C).
A TAR DNA mutant for which all four internal loops were deleted (-L4-L3-L2-L1, Fig. 1 E) is characterized by a fully closed conformation with or without NC, Eappmol 0.97, Eapp = 0.04 (Fig. 2, D and F, respectively). In fact, the individual Eapp(t) trajectories for this TAR DNA mutant with or without NC are indistinguishable from trajectories for -L4-L3 mutant hairpins without NC (Fig. 2 E). This result confirms previous reports on the critical importance of internal bulges in the hairpin structure to observe an NC effect (Beltz et al., 2003).
The data provide direct evidence at the individual hairpin level that NC activity in TAR DNA is limited to internal loops L1 and L2 and does not involve the internal bulges L3 and L4 (see Scheme 1). Thus, all hairpin constructs conserving the two internal loops L1 and L2 have identical distributions of Eapp(t) when complexed with NC, irrespective of the presence of other destabilizing internal bulges. In comparison, the hairpin conserving only the last internal loop can only access a conformation with a smaller end-to-end distance when complexed with NC. Finally, the construct where all the internal loops and bulges have been deleted is only found in the closed conformation.
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Care has been taken to rule out any influence that fluorophore blinking (reversible photobleaching) or fluorophore quenching by NC might have on the observed trajectories and distributions of Eapp(t). Thus, we note that no emission quenching is observed upon NC addition to the construct that lacks internal loops and bulges. On the other hand, Cy5 blinking events were removed from the Eapp(t) trajectories as described in the Experimental Setup section. We conclude that the Eappmol < 0.97 observed in hairpins with internal loops/bulges (Fig. 2, A–C) in equilibrium with NC is a direct result of NC-hairpin interactions.
Consistent with a closed hairpin structure, the distribution of Eapp(t) values for -L4-L3 mutant single molecules in the absence of NC has a mean value Eappmol = 0.97, Eapp = 0.04, (Fig. 2 E). Fig. 3 portrays the normalized ensemble distributions of Eapp(t) for the different hairpins with and without NC to facilitate their comparison.
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). From the cross-correlation analyses we were able to assign the rate constants associated with the observed hairpin conformational distributions.
The correlation analysis of the data reveals dynamics in the milliseconds time domain for hairpins with one or more internal bulges in equilibrium with NC. Consistent with dynamics reflecting FRET efficiency fluctuations, donor and acceptor intensities are anticorrelated; i.e., an increase in donor intensity is accompanied by a decrease in acceptor intensity (Kim et al., 2002). Cross correlations were converted to Eapp autocorrelations as described in the Experimental Setup section.
Table 1 lists the relaxation rate constants and amplitudes derived from the cross-correlation analysis of IA(t) and ID(t) trajectories for TAR DNA/NC, -L4-L3 mutant/NC, and -L4-L3-L2 mutant/NC (see columns 1 and 2). A single exponential relaxation with a rate constant kr = 4 x 102 s–1 was measured for -L4-L3-L2 mutant/NC. The single exponential decay indicates that this is a two-state transition between an open and a closed hairpin conformation. The Eapp autocorrelation relaxation rate constants for TAR DNA/NC and for -L4-L3 mutant/NC have values of kr = 3 x 102 s–1 and 2 x 102 s–1, respectively. Conformational equilibration (relaxation) occurs on a timescale of a few milliseconds, much shorter than the observation time (4–20 s) for each hairpin, ensuring equilibrium sampling in the observed single molecule data.
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). The predominantly open hairpin conformation directly observed from the distribution of Eapp(t) reveals that for TAR DNA/NC or -L4-L3 mutant/NC the opening rate constant is much larger than the closing rate constant. As much as 10% of the Eapp events can be assigned to a closed hairpin conformation. In a two-state model this would indicate that opening and closing rate constants for TAR DNA/NC or -L4-L3/NC are in the range between kclosing 
3 x 101 s–1 and kopening 2.5 x 102 s–1.
In an attempt to shift the equilibrium from the predominantly open hairpin conformation directly observed from the distribution of Eapp(t) for TAR DNA/NC and -L4-L3 mutant/NC we increased the MgCl2 concentration by 10-fold. Fig. 4 illustrates the distribution of Eappmol values (left) and a representative single molecule Eapp(t) distribution (middle) and time trajectory (right) in the presence of 2.5 mM MgCl2. Consistent with a stabilization of the closed hairpin by MgCl2 (Misra and Draper, 1998) the equilibrium was shifted to the closed conformation after addition of 2.5 mM MgCl2. Under these conditions the measured relaxation rate constant is 0.7 x 102 s–1 and the closed-open equilibrium constant is Kclosed-open 1. If a two-state model applies to these conditions opening and closing rate constants for -L4-L3 mutant/NC are kclosing kopening = 0.4 x 102 s–1. Two distinct peaks at 0.8 and 0.97 were resolved in the distribution of Eapp(t). These observations support the conclusion that the dynamics of hairpin opening-closing relaxation in the presence of NC occur in the millisecond time domain.
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; Beltz et al., 2003). The latter study was carried out using fluorescence correlation spectroscopy (FCS), a technique that is unable to measure slow dynamics, using a related cTAR DNA and truncated NC system. To determine if faster events escaped our detection, we cross correlated the donor and acceptor intensities of single immobilized hairpins over a time range from 0.5 µs to 4 s with an ALV5000/E correlator board. Fig. 5 portrays the ensemble Eapp autocorrelation derived from donor-acceptor cross correlations. No fast components are observed in -L4-L3-L2 mutant/NC for which the relaxation rate is similar to that obtained from the Eapp(t) trajectories (Table 1; compare columns 2 and 4). The relaxation for TAR DNA/NC and -L4-L3 mutant/NC spanned from microseconds to milliseconds with approximately equal weights in both time domains. A biexponential fit to the data generates relaxation rate constants of 2 x 102 s–1 and 3 x 103 s–1 for TAR DNA/NC and 2 x 102 s–1 and 2 x 103 s–1 for -L4-L3 mutant/NC, respectively (Table 1; columns 4 and 6). In the presence of 2.5 mM MgCl2, the decay for -L4-L3 mutant/NC is biexponential with relaxation rate constants of 0.8 x 102 s–1 and 6 x 102 s–1.
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We performed control experiments to determine the effect, if any, that Cy5 photophysical properties have on the correlation curves measured (Widengren et al., 2001; Widengren and Schwille, 2000). Experiments were done with the -L4-L3 mutant in the absence of NC and with the -L4-L3-L2-L1 mutant in the absence and presence of NC. The cross correlation for the controls had an amplitude 0 after 2 x 10–4 s. There were no donor-acceptor anticorrelated fluctuations with lifetimes of 100 µs or longer. Cross correlations at times < 2 x 10–4 s appear to be dominated by Cy5 cis-trans isomerization, unrelated to FRET dynamics. A more complete discussion of the early time behavior of the autocorrelation is given in the Supplementary Material.
There is no measurable change in the single molecule data when BSA is replaced by an alternative surface coating, polyethyleneglycol (PEG). This strongly suggests that surface interactions are not significant because these surfaces are known to present very different chemical environments. The Eapp(t) distribution mean value and standard deviation and the Eapp autocorrelation amplitudes and relaxation rate constants are the same within experimental error for PEG and BSA. In particular, results on the effect of NC complexation on the TAR DNA hairpin end-to-end dynamics and end-to-end equilibrium distribution obtained with polyethyleneglycol (MW 2000, Nektar Therapeutics, Huntsville, AL) and biotinylated polyethyleneglycol (MW 5000, Nektar Therapeutics) coated coverslips (see Fig. S7 in Supplementary Material) (Ha, 2001; Rasnik et al., 2004) are identical to those obtained with the BSA-based immobilization scheme reported here.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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1 ms) much shorter than the observation time (4–20 s) for each hairpin insuring equilibrium sampling in the observed single molecule data. It can be argued that the observed partially open TAR DNA/NC conformation is a critical intermediate in the NC catalyzed annealing mechanism of TAR DNA/RNA, because the open terminal stems possess 21 unpaired bases that are accessible for DNA/RNA annealing. |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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This research was supported by National Institutes of Health (NIH) grant AI43231 (K.M.F.), NIH grant GM65818 (P.F.B.), and NIH postdoctoral National Research Service Award grant F32 AI10463 (E.J.H.).
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FOOTNOTES |
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Submitted on March 18, 2004; accepted for publication June 17, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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