Single-stranded Polynucleotides Research Articles

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme.

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.

Three‐way multivariate curve resolution applied to speciation of acid–base and thermal unfolding transitions of an alternating polynucleotide

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.

Stretching Single-Stranded DNA: Interplay of Electrostatic, Base-Pairing, and Base-Pair Stacking Interactions

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.

Novel DNA-Polysaccharide Triple Helixes and Their Application to a Gene Carrier

Schizophyllan is a polysaccharide that belongs to the β-(1 → 3)-glucanfamily and has been known to activate the immunity system. This paper reviews our recent finding that schizophyllan can form a macromolecular complex with single-stranded polynucleotides and this complex is applicable to a new class of gene carriers.

Three-way multivariate curve resolution applied to speciation of acid-base and thermal unfolding transitions of an alternating polynucleotide

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.

Mutational analysis of hepatitis C virus NS3-associated helicase.

Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational-functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase-ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr(392) and Trp(501) with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.

Structure-based mutagenesis study of hepatitis C virus NS3 helicase.

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides.

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of gamma-interferon (gammaIFN), i.e., ds polynucleotides increase class I much more than class II, whereas gammaIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-kappaB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from gammaIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

The Binding Affinity of Ff Gene 5 Protein Depends on the Nearest-Neighbor Composition of the ssDNA Substrate

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Kω (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Kω values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA 48, dC 48, d(AAC) 16, d(ACC) 16, d(AACC) 12, and d(AAACC) 9A 3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.

Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase

Synthesis of deoxynucleotide-containing RNA-like single-stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8–10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1–+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG-containing) was achieved with high yields.

Bluetongue virus core protein VP4 has nucleoside triphosphate phosphohydrolase activity.

The intact virion of bluetongue virus comprises ten segments of dsRNA enclosed in two concentric protein capsids. The core, which is transcriptionally active, includes three minor proteins (VP1, VP4 and VP6) which are considered to be the candidates for the core-associated enzymes that transcribe and modify full-length mRNA copies for each of the ten genome segments. Using purified recombinant VP4 protein and core-like particles containing VP4, in this report it is demonstrated that VP4 has nucleoside triphosphatase (NTPase) activity. VP4 is a nonspecific NTPase that hydrolyses four types of ribonucleoside triphosphate (NTP) to the corresponding nucleoside diphosphate. The substrate preference was GTP>ATP>UTP>CTP. NTP hydrolysis by VP4 was maximal when the Mg2+ or Ca2+ ion concentrations were 4 mM or 6 mM, respectively. The presence of single-stranded polynucleotides poly(A), poly(U) and poly(C) had little effect on the NTPase activity. Although the enzyme exhibited a broad temperature optimum around 40 degrees C, the pH optimum was sharp, between pH 7.5 and 8. The Km and Vmax of ATP hydrolysis were calculated to be 0.25+/-0.05 microM ATP and 55+/-4 pmol ATP hydrolysed min(-1) microg(-1), respectively. The Km was affected by the addition of poly(A) to only a small extent in contrast to the Vmax, which was increased by at least twofold.

Displacement of viral DNA termini from stable HIV-1 integrase nucleoprotein complexes induced by secondary DNA-binding interactions.

The human immunodeficiency virus type-1 (HIV-1) integrase is known to form a highly stable interaction with the termini of the linear, pre-integrated retroviral genome, where it catalyzes the 3'-OH processing and strand transfer processes required for their coordinated integration into host DNA. Here, we determine that the association of HIV-1 integrase with the viral DNA termini leads to the formation of two classes of nucleoprotein complexes with distinct properties in vitro. Both bound states are intrinsically stable and highly resistant to exonuclease digestion, but nonetheless they exhibit different stabilities in the presence of single-stranded polynucleotides. While a population of preassembled complexes tolerates elevated polynucleotide concentrations, the remainder forms an unstable ternary (integrase-substrate-polynucleotide) intermediate, leading to the rapid expulsion of the otherwise tightly bound substrate. The distribution of complexes between the two states is influenced by the preincubation time and temperature, increases in either of which favor the formation of the challenge-resistant species. Challenge-resistant complexes are formed more efficiently with Mn2+ than with Mg2+ and are sensitive to the length rather than the sequence of the DNA substrate. Due to the delayed appearance of the challenge-resistant form after the initial stable binding of the DNA substrate, our results may be indicative of a structural change in the preassembled complex which thereby modulates its response to exogenous DNA targets.

Error Analysis of Chemically Synthesized Polynucleotides

Two single-stranded polynucleotide constructs, 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long were prepared from the polynucleotides by primer extension with a DNA polymerase and end-trimming with two restriction endonucleases, then the fragments were ligated into separate plasmids. Errors in individual insert copies were determined by dideoxy sequencing after in vivo amplification of plasmids. Five of the ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion and one G-->C transversion. The origins of the latter two errors are unclear, but single-base deletions are inconsistent with errors of polymerases; thus, the most common sequence errors of chemical synthesis are deletion mutations. Deletions are most likely to result from incomplete capping or de-tritylation. The observed error rate can became a significant limiting factor in applications that depend on the correctness of a polynucleotide sequence in individual insert clones.

Interactions of the bacteriophage T4 gene 59 protein with single-stranded polynucleotides: binding parameters and ion effects

The gene 59 protein (gp59) of bacteriophage T4 is an important accessory protein of the phage-encoded replicative DNA helicase, gp41. The properties of this 26 kDa protein include selective binding to ssDNA, and specific interactions with both gp41 and gp32, the T4-encoded ssDNA-binding protein. gp59 stimulates many of the DNA-dependent activities of the gp41 enzyme by promoting its assembly onto gp32-ssDNA complexes. Direct interactions between gp59 and gp32-ssDNA complexes are essential for helicase assembly, and gp59-gp32 protein-protein interactions have been shown to play a central role. Presumably, the ssDNA-binding activity of gp59 is also important for helicase assembly; however, to date this activity has been poorly characterized. In this study, we present the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluorescence enhancement and quantitative ssDNA-cellulose methods, we demonstrate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monomer; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > εDNA (random sequence) > poly(dA) > poly(dεA); (3) gp59 exhibits two or more different polynucleotide binding modes distinguished by their cooperativities of binding, and modulated by salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with multiple ionic contacts between protein and ssDNA phosphate residues and with the displacement of anions from the protein. The implications of our findings for the mechanism of action of gp59 in helicase-ssDNA assembly are discussed.

Electrochemical analysis of formation of polynucleotide complexes in solution and at electrode surfaces

The formation of double- and triple-stranded complexes between the synthetic homopolyribonucleotides in solution and at the surfaces of the hanging mercury drop (HMDE) and carbon paste (CPE) electrodes was studied by means of voltammetric and chronopotentiometric methods. It was shown that the cyclic voltammetry reduction signal of poly(C) and the anodic signal of poly(G) obtained with HMDE, as well as the chronopotentiometric oxidation signal of poly(G) obtained with CPE can be used to detect the duplex and triplex formation in solution. In addition to these intrinsic polynucleotide signals, the indicator [Co(phen) 3] 3+ was used to differentiate between single-stranded and ordered structures at CPE. It was shown that the indicator binds preferentially to the duplex poly(G) · poly(C) (formed in solution followed by adsorption at CPE) displaying, however, no preferential binding to the triplex structures. If poly(C) or poly(G) were immobilized at the electrode surface (by adsorption forces) and hybridized with the complementary polynucleotide in solution (at neutral pH), only a small extent of complexation could be detected at HMDE under the given conditions. On the other hand, analogous experiments with CPE suggested formation of ordered polynucleotide structures at the carbon electrode surface. As a result of complexation of poly(C) with poly(G) at neutral pH, formation of more than one ordered structure, including the duplex, was indicated. Polynucleotide complexation performed at pH 3.3 (optimum for formation of the protonated triplex poly(C +) · poly(G) · poly(C) in solution) resulted in the formation of a mixture of structures including triplex and duplex structures. Binding of [Co(phen) 3] 3+ to the product of poly(A) complexation with poly(U) suggested that the yield of the duplex formed at the CPE surface may be influenced by the conformation of the single-stranded polynucleotide anchored at the surface prior to the hybridization. The implications of these findings to DNA biosensor development are discussed.

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the G · C sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the A · T and A · U sequences. G · C base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to A · T base pairs. On the contrary, the continuous ribonucleotide G · C sequence binds the phenazines more weakly than does the A · U sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.

Single-stranded DNA binding properties of the uvsy recombination protein of bacteriophage T4

The uvsY protein is an essential component of the bacteriophage T4 general recombination machinery. The properties of this 16 kDa protein include selective binding to ssDNA, as well as specific protein-protein interactions with other T4 recombination proteins including uvsX (general recombinase) and gp32 (ssDNA-binding protein). uvsY promotes the assembly of uvsX-ssDNA filaments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA. To better understand the role of uvsY in the T4 recombination system, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model single-stranded polynucleotide, εDNA, which is a fluorescent, etheno-modified form of random-sequence ssDNA. We have found that the binding of uvsY protein enhances the fluorescence of the εDNA lattice and that the maximal amount of fluorescence enhancement observed is dependent on salt concentration. In addition, we have used the εDNA fluorescence enhancement assay to establish thermodynamic parameters of binding and to define some of the molecular details of uvsY-εDNA interactions. We show that uvsY binds to εDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to εDNA over unmodified ssDNA. The significance of these results is discussed in light of current models of uvsY action in the T4 recombination system.

The Role of Hydrophobic Versus Hydrophilic Base Character in the Anti-HIV Activity of Purine-Containing Polyribonucleotides

ABSTRACT In contrast to the highly amphiphilic poly(1-methyl-6-thioinosinic acid), a potent anti-HIV agent, poly(1-amino-6-thioinosinic acid) (PATI) lacks the unique melting behavior characteristic of the amphiphilic polymers and is completely devoid of anti-HIV activity. This is consistent with the hypothesis that amphiphilic character and the ability to form an ordered secondary structure in solution are prerequisites for potent anti-HIV activity of single-stranded polynucleotides.

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.