Solution Structure of Poly(dA-dT)· Poly(dA-dT) in Low and High Salt: A 500 MHz 1H NMR Study Using One-Dimensional NOE

Abstract

CD spectra of poly(dA-dT)· poly(dA-dT) in low salt (10–100 mM NaCl) and high salt (4–6 M CsF) are different i.e. 275 nm band gets inverted in going from low to high salt (Vorhickova et. al.MarJ. Mol. Biol. 166, 85, 1983). However, from CD spectra alone it is not possible to decipher any structural differences that might exist between the low and high salt forms of poly(dA-dT)? poly(dA-dT). Hence, we took recourse to high resolution NMR spectroscopy to understand the structural properties of poly(dA-dT)? poly(dA-dT) in low and high salt. A detailed analysis of shielding constants and extensive use of NOE studies under minimum spin diffusion conditions using C(8)-deuterated poly(dA-dT)? poly(dA-dT) enabled us to come up with the following conclusions (i) base-pairing is Watson-Crick under low and high salt conditions, (ii) under both the conditions of salt the experimental data can be explained in terms of an equilibrium blend of right and left-handed B-DNA duplexes with the left-handed form 70% and the right-handed 30%. In a 400 base pairs long poly(dA-dT)? polyidA-dT) (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis, (iii) However, there are other structural differences between the low and high salt forms of poly(dA-dT) ? poly(dA-dT); under the low salt condition, right-and left-handed B-DNA duplexes have mononucleotide as a structural repeat while under the high salt conditions, right-and left-handed B-DNA duplexes have dinucleotide as a structural repeat. In the text we provide the listing of torsion angles for the low and high salt structural forms, (iv) Salt (CsF) induced structural transition in poly(dA-dT)? poly(dA-dT) occurs without any breakage of Watson- Crick pairing, (v) The high salt form of poly(dA-dT)? poly(dA-dT) is not the left-handed Z-helix.

Although the results above from NMR data are quite unambiguous, a question still remains i.e. what does the salt (CsF) induced change in the CD spectra of poly(dA-dT)? poly(dA-dT) really indicate? Interestingly, we could show that the salt (CsF) induced change in poly(dA-dT)? poly(dA-dT) is quite similar to that caused by a basic polypeptide viz. poly-L(Lys₂-Ala)_n i.e. both the agents induced a ψ-structure in DNA. And it was also demonstrated that the changes in poly(dA-dT)? poly(dA-dT) as caused by CsF and poly-L-(Lys₂-Ala)_n could be reverted back by ethidium bromide-a relaxing agent.

To minimize complications from spin diffusion in this study we have used very small presaturation pulse lengths and C(8)-deuterated poly(dA-dT)? poly(dA-dT) of 400 ± 150 bp long. Even though deuteration of a primary site of diffusion such as C(8)H substantially decreases diffusion, in order to make sure that our conclusions are not compromised by possible diffusion in such a long fragment under small presaturation times, we have repeated our experiments using the six base pair long duplex of d(A-T-A-T-A-T) and found the results to be strikingly similar to that from the polymer.     

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dA-dT) poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni²⁺ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl₂ in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm″^?1coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm^?1 region characterizing local chemical interactions of the Ni²⁺ ions with the adenien N7 position, iii) lines at about 1483-and 1582 cm^?1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680-and 1733 cm^?1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT) poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Conformational Variability of Poly(dA-dT)·Poly(dA-dT) and Some Other Deoxyribonucleic Acids Includes a Novel Type of Double Helix

Abstract

The article reviews data indicating that poly(dA-dT)?poly (dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT)?poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the ³¹P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the ³¹P NMR spectrum of the limiting alternating B conformation of poly(dA-dT)?poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack.

However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the ³¹P NMR resonance of poly (dA-dT)?poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC)?poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC) ?poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT)?poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT)?poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT)?poly(dA-dT) and poly(dG-dC)?poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT)?poly(dA-dT) X-DNA.

It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA. These indicate that Arich regions in natural DNAs can isomerize into the X form while the bulk of the molecule remains in the B form. The coexistence of both structures in a single DNA molecule may be understood in view of the favourable kinetic and thermodynamic properties with which the X form appears.     

Structure and Dynamics of Netropsin-Poly(dA-dT)· Poly(dA-dT) Complex: 500 MHz 1H NMR Studies

Abstract

Antibiotic netropsin is known to bind specifically to A and T regions in DNA; the mode of binding being non-intercalative. Obviously, H-bonding between the proton donors of netropsin and acceptors N3 of A and 02 of T comes as a strong possibility which might render this specificity. In netropsin there could be 8 proton donors: four terminal amino groups and four internal imino groups. However, methylation of the terminal amino groups does not alter the binding affinity of netropsin to DNA—but the modification of the internal imino groups significantly lowers the binding affinity. Hence, the logical conclusion is that netropsin may specifically interact with A and T through H-bonding and in order to do so, it should approach the helix from the minor groove. The present paper provides experimental data which verify the conclusion mentioned above.

Using poly(dA-dT)? poly(dA-dT) as a model system it was observed following a thorough theoretical stereochemical analysis that netropsin could bind to -(T-A-T) sequence of the polymer in the B-form through the minor groove by forming specific B-bonding. Models could be either right or left-handed B-DNA with a mono or dinucleotide repeat.

By monitoring the ³¹P signals of free poly(dA-dT) ? poly(dA-dT) and netropsin-poly(dA-dT)? poly(dA-dT) complex we show that the drug changes the DNA structure from essentially a mononucleotide repeat to that of very dominant dinucleotide repeat; however the base- pairing in the DNA-drug complex remain to be Watson-Crick. Whether H-bonding is the specific mode of interaction was judged by monitoring the imino protons of netropsin in the presence of poly(dA-dT) ? poly(dA-dT). This experiment was conducted in 90% H₂O + 10% D₂O Using the time-shared long pulse. It was found that exchangeable imino protons of netropsin appear in the drug-DNA complex and disappear upon increasing the D₂O content; thus confirming that H-bonding is indeed the specific mode of interaction. From these and several NOE measurements, we propose a structure for poly(dA-dT)? poly(dA-dT(-netropsin complex.

In summary, experimental data indicate that netropsin binds to poly(dA-dT)? poly(dA-dT) by forming specific hydrogen bonds and that the binding interaction causes the structure to adopt a Watson-Crick paired dinucleotide repeat motif. The proposed hydrogen bonds can form only if the drug approaches the DNA from the minor groove. Within the NMR time scale the interaction between the ligand and DNA is a fast one. From the NOE experimental data, it appears that poly(dA-dT)? poly(dA-dT) in presence of netropsin exists as an equilibrium mixture of right- and left-handed B-DNA duplexes with a dinucleotide repeat—with a predominance of the left-handed form. The last conclusion is a soft one because it was very difficult to make sure the absence of spin diffusion. In a 400 base pairs long DNA duplex- drug complex (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis.     

Two Binding Modes of Netropsin are Involved in the Complex Formation with Poly(dA-dT)·Poly(dA-dT) and other Alternating DNA Duplex Polymers

Abstract

Using CD measurements we show that the interaction of netropsin to poly(dA-dT)·poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA·dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA)·poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT)·poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

Description of Base Motion and Role of Excitations in the Melting of Poly(dG) · Poly(dC)

Abstract

We study the contribution of various vibrational modes to the melting of poly(dG) · poly(dC). We find that the principal contribution comes from the H-bond breathing modes that have been observed in Raman scattering and that we have associated with helix melting. We show the softening of these modes on approach to melting in agreement with the observed behavior. We also describe the contribution to melting from base rotation modes that others have suggested are important in melting.     

Poly(ADP-Ribose)—A Unique Natural Polymer Structural Features,Biological Role and Approaches to the Chemical Synthesis

Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them, poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective target for the design of new drugs, affecting PAR metabolism. The structure of PAR was established by enzymatic hydrolysis and further analysis of the products, but total chemical synthesis of PAR hasn't been described yet. Several approaches have been developed on the way to chemical synthesis of this unique biopolymer.     

Polarized Micro Raman Spectroscopic Studies of Nucleic Acid: Local Raman Tensors in Inosine-5′-monophosphoric Acid and the Orientation of Ekypoxanthine Residue in Poly(rI). Poly(rC) Fiber

Abstract

The polarized Raman spectra of a single crystal of the barium salt of inosine monophosphoric acid hexahydratc (Ba-IMP.6H₂O) have been observed with 488.0 nm excitation. For each Raman band, the relative intensities of aa, bb, cc, ab and bc tensor components have been determined. The tensor quotients from the crystal were augmented with measured depolarization ratios in solution. From these experimental data, the shape and orientation of the localized Raman scattering tensor were deduced for each of the normal modes of the hypoxanthine residue, phosphate moiety and ribose portion. The hypoxanthine residue gives a strong Raman band around 1553 cm^?1, which shows rather large depolarization ratio, p = 0.32, in aqueous solution, and shows a great scattering anisotropy in the single crystal of IMP. The shape and orientation of the Raman tensor associated to this 1553 cm^?1 vibration have been determined: one of its principal axes (y-axis) is directed along the long axis (N1-N7) of the hypoxanthine residue and the relative magnitudes of its components are given as r ₁ = α_xx/α_zz = ?1, r ₂ = α_yy/α_zz = 12. Next, a general relation has been shown between the orientation angles (θ and χ) of such a local Raman tensor in a uniaxial biological fiber and the anisotropy of Raman scattering intensities from the fiber. By the use of this relation, a discussion has been made of the orientation of the hypoxanthine residue in a poly(r1). poly(rC) duplex fiber.

     

The Complex of Poly(dG) · Poly(dC) with Arginine: Stabilization of the B Form and Transition to Multistranded Structures

Abstract

We have studied by X-ray diffraction fibers of complexes of poly(dG)·poly(dC) with N-α-acetyl-L-arginine ethylamide. Although these polynucleotides favour the A form of DNA, in this complex it is never found, thus confirming that arginine prevents the appearance of this form of DNA At high relative humidity the B form is present. Upon dehydration two new structures appear. One of them is a triple helix, most likely formed by poly(dC⁺) · poly(dG) · poly(dC). The other structure found also has features which indicate a multistranded conformation.     

Raman Spectroscopy Study of the B-Z Transition in (dG-dC)n · (dG-dC)n and a DNA Restriction Fragment

Abstract

The B to Z conformational transition of (dG-dC)_n·(dG-dC)_n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831–833 and 1093 cm^?1. Both (dG-dC)_n·(dG-dC)_n and the 157 bp DNA show changes in the guanine vibration at 682 cm^?1 and backbone band at 831–3 cm^?1 preceeding a highly cooperative change in the 1093 cm^?1 PO 7 vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway.

We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)_n·(dG- dC)_n indicate that in 4.5 M NaC/the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.     

Use of a combination of the RDC method and NOESY NMR spectroscopy to determine the structure of Alzheimer’s amyloid Aβ10–35 peptide in solution and in SDS micelles

The spatial structure of Alzheimer’s amyloid Aβ_10–35-NH₂ peptide in aqueous solution at pH 7.3 and in SDS micelles was investigated by use of a combination of the residual dipolar coupling method and two-dimensional NMR spectroscopy (TOCSY, NOESY). At pH 7.3 Aβ_10–35-NH₂ adopts a compact random-coil conformation whereas in SDS micellar solutions two helical regions (residues 13–23 and 30–35) of Aβ_10–35-NH₂ were observed. By use of experimental data, the structure of “peptide–micelle” complex was determined; it was found that Aβ_10–35-NH₂ peptide binds to the micelle surface at two regions (residues 17–20 and 29–35).     

18O-Labeled Adenosine and 9-(β-D-Arabinofuranosyl)Adenine (ARA-A): Synthesis,Mass Spectrometry,and Studies of 18O-Induced 13C NMR Chemical Shifts

Abstract

[2′-¹⁸O]- and [3′-¹⁸O]-Adenosine and [2′-¹⁸O]- and [3′-¹⁸O]-9-(β-D-arabinofuranosyl) adenine were synthesized from^?appropriate nucleoside precursors. The sites of ¹⁸O-incorporation were determined by mass spectrometry. ¹⁸O-Induced ¹³C NMR shifts were measured for 2′-and 3′-labeled adenosines as 1.2 and 1.6 Hz, respectively.     

DFT study of (17)O, (1)H and (13)C NMR chemical shifts in two forms of native cellulose, I(α) and I(β)

This computational study is intended to shed light on the crystalline and molecular structure, together with the hydrogen bonding (H-bonding) differences between two forms of native cellulose. DFT calculations were carried out to characterize the ¹⁷O, ¹H and ¹³C nuclear magnetic resonance (NMR) parameters in cellulose I_α and I_β with the B3LYP functional employing the 6–311++G7 and 6–31+G1 basis sets. Geometry optimization revealed that the average HB length is shortened by 0.01–0.08 Å when the chains are aligned, whereas the average bond angle increases by about 4–8° exhibiting the enhancement of HB strength. For the isolated cellotetramer chains, the isotropic ¹⁷O–H chemical shifts were plotted as a function of HB length. Our results indicated that as the HB length in cellotetramer I_α increases, the ¹⁷O–H chemical shift isotropy increases, but this parameter changes in the opposite direction for the other structure. Moreover, B3LYP/6–311++G7 calculations reveal that there is an acceptable correlation between the calculated ¹³C chemical shifts of the two structures and their experimental values.     

Application of Abscisic Acid (S-ABA) to ‘Crimson Seedless’ Grape Berries in a Mediterranean Climate: Effects on Color, Chemical Characteristics, Metabolic Profile, and S-ABA Concentration

‘Crimson Seedless’ is a table grape cultivar that often fails to develop adequate red color in Mediterranean climates. Application of abscisic acid (S-ABA) may be an aid for improving color, but its potential effects on overall quality and S-ABA concentration of the berry should be also considered. We tested two concentrations (200 and 400 mg/L) and different times of application (from 1 week after veraison up to 9 days before harvest) of a commercial formulation of S-ABA (ProTone^®) to verify the effect on harvestable bunches, color, chemical characteristics, metabolic profile, and S-ABA concentration in the berry. It was found that either the application of S-ABA at 400 mg/L one week after veraison or the application of S-ABA at 400 mg/L one week and four weeks after veraison positively affected the berry skin color, shifting the hue (h°) from 20 to a more red-violet hue (h° = 11–12). In general, the application of S-ABA, with the exception of the late treatments, enhanced coloration of the berries and increased the amount of harvestable bunches at the first pick because it promoted the skin-coloring process. S-ABA did not affect berry firmness but reduced the berry detachment force. Nevertheless, the values remained sufficiently high and the general quality of the bunch was not compromised. Ripening parameters (°Brix, pH, titratable acidity) were not affected by S-ABA applications, and even the primary metabolite profile was not influenced by the treatments as ascertained by multivariate statistical analyses [principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA)] applied to nuclear magnetic resonance (NMR) data. The S-ABA concentration in the berry, when treatments were performed around veraison, was within the natural range for grape (10–400 ng/g f.w.), whereas when late treatments were applied (few days before harvest), the concentration was higher (more than 1,000 ng/g f.w.). The best results for yield, quality, and S-ABA concentration in the berry were observed for the treatments performed a few days after veraison at the dose of 400 mg/L. This study gives new information about the positive effects of S-ABA on color without any particular change in the metabolic profile of the berry.     

Use of immobilized Streptomyces griseus proteases (pronase) as a probe of structural transitions of lysozyme, β-lactoglobulin and casein

The native - denatured (N U) structural transition in lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), β-lactoglobulin and caseins have been studied by proteolysis using immobilized Streptomyces griseus proteases (pronase) as a probe. A diverse range of susceptibility to urea denaturation was revealed by evaluation of initial rates and pseudo first-order rate constants for hydrolysis of these proteins. Comparison of the rate of hydrolysis of lysozyme vis-à-vis performic acid oxidized-lysozyme showed that the degree of backbone accessibility for native lysozyme, even in concentrated urea solutions, was less than that of the oxidized protein. At pH 7.5, native lysozyme appeared to possess the most stable structure, followed by β-lactoglobulin and, finally, the caseins. It is postulated that the proteolytic rate depends upon accessibility of a susceptible bond(s) or subtle conformational changes in the least stable domain. Following cleavage of this bond(s), K_D increases thus exposing more backbone. Use of pronase immobilized on porous succinamidopropyl-glass beads resulted in increased enzyme stability and eliminated autolysis. Consequently, immobilized proteases are an excellent probe of structural transitions of protein substrates in denaturants.     

Plasma anti-α-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a)

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O–glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O–glycan-specific lectin jacalin. O–Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.     

Associations of aquatic insects (Ephemeroptera,Plecoptera, and Trichoptera) in a network of subarctic lakes and streams in Québec

As part of an impact assessment of large hydroelectric projects in the James Bay drainage in Northwestern Québec, the aquatic insect communities were studied in a network of rivers, lakes and streams during the summer of 1975. Thirty-eight emergence traps operated over the ice-free season yielded 10 888 insects (5559 Ephemeroptera, 2817 Plecoptera, and 2512 Trichoptera), representing 148 species (respectively 44, 18, and 86), most of temperate and boreal afinities. There was no arctic element.Similarity analyses and clustering procedures on the emergence series revealed the existence of distinct insect communities in the river (fast and slow sections), the streams (fast and slow), the lakes and the bogs, each characterized by a particular assemblage of species. Many of the species were more or less ubiquitous and differences between communities were marked more by changes in the dominance of the species and differences in the frequency distributions, than absolute shifts in the species lists. The yields in the traps set in fast water were much greater than those in slow running water, and these in turn greater than those of standing water.By comparison with more southerly sites, the seasonal succession of species was retarded in the spring and early summer, but was not shortened appreciably in the fall. The usual emergence patterns associated with these taxa was observed, namely those of spring, summer and autumn species.     

Multistrain Probiotic Modulation of Intestinal Epithelial Cells' Immune Response to a Double-Stranded RNA Ligand,Poly(I·C)

A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both T_H1 and T_H2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3 [TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(I·C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(I·C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory T_H1 and antiviral innate immune responses compared to that obtained by the poly(I·C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3 domain-containing adapter-inducing beta interferon (TRIF), mitogen-activated protein kinase, and NF-κB signaling pathways. Such a model may be useful for selecting potential biomarkers for the design of future clinical trials.