Intermolecular Duplex Research Articles

Mechanistic Insights into the Kinetics of HIV-1 Nucleocapsid Protein-facilitated tRNA Annealing to the Primer Binding Site

HIV-1 reverse transcriptase uses human tRNALys,3 as a primer to initiate reverse transcription. Prior to initiation, the 3′ 18 nucleotides of this tRNA are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). Here, we show that the HIV-1 nucleocapsid protein (NC) enhances this annealing by approximately five orders of magnitude in vitro, decreasing the transition state enthalpy from approximately 20kcalmol−1 for the uncatalyzed reaction to 13kcalmol−1 for the NC-catalyzed process. Moreover, the annealing follows second-order kinetics, consistent with the nucleation of the intermolecular duplex being the rate-limiting step. This nucleation is preceded by melting of a small duplex region within the original structure, and is followed by much faster zipping of the rest of the 18 base-pair (bp) duplex. A tRNA mutational analysis shows that destabilization of the tRNA acceptor stem has only a minor effect on the annealing rate. In contrast, addition of bases to the 5′ end of tRNA that are complementary to its single-stranded 3′ end interferes with duplex nucleation and therefore has a much larger effect on the net reaction rate. Assuming that the apparent transition free energy of the annealing reaction, ΔG‡, is a sum of the melting (ΔGm) and nucleation (ΔGnuc) free energies, we show that NC affects both ΔGm and ΔGnuc. We estimate that ten to 100-fold of the overall rate enhancement is due to NC-induced destabilization of a 4 bp helix in the PBS, while the additional factor of 103–104 rate enhancement is a result of NC-facilitated duplex nucleation. The apparently similar effectiveness of wild-type and SSHS NC, a mutant that lacks the zinc finger structures, in facilitating the tRNA annealing reaction is most likely the result of the mutual cancellation of two factors: SSHS NC is less effective than wild-type NC as a duplex destabilizer, but more effective as a duplex nucleating agent.

Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase.

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3'-ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3'-end complementarity, therefore yielding an RNA-primed RNA-template. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3'-end, to produce RNA transcripts theoretically unable to form 3'-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions.

In Vitro Evidence That the Untranslated Leader of the HIV-1 Genome Is an RNA Checkpoint That Regulates Multiple Functions through Conformational Changes

The HIV-1 RNA genome forms dimers through base pairing of a palindromic 6-mer sequence that is exposed in the loop of the dimer initiation signal (DIS) hairpin structure (loop-loop kissing). The HIV-1 leader RNA can adopt a secondary structure conformation that is not able to dimerize because the DIS hairpin is not folded. Instead, this DIS motif is base-paired in a long distance interaction (LDI) that extends the stem of the primer-binding site domain. In this study, we show that targeting of the LDI by either antisense oligonucleotides or specific mutations can induce the conformational switch to a branched multiple hairpin (BMH) structure, and this LDI-to-BMH switch coincides with increased RNA dimerization. Another interesting finding is that the extended LDI stem can resist a certain level of destabilization, indicating that a buffer is created to prevent a premature conformational switch and early dimerization. Because the tRNA(Lys3) primer for reverse transcription anneals to multiple sequence elements of the HIV-1 leader RNA, including sequences in the LDI stem, we tested whether tRNA-annealing can destabilize the LDI stem such that RNA dimerization is triggered. Using a combination of stem-destabilizing approaches, we indeed measured a small but significant effect of tRNA-annealing on the ability of the RNA template to form dimers. This observation suggests that HIV-1 RNA can act as a checkpoint to control and coordinate different leader functions through conformational switches. This in vitro result should be verified in subsequent in vivo studies with HIV-infected cells.

Binding of oligonucleotides to a viral hairpin forming RNA triplexes with parallel G*G*C triplets

Infrared and UV spectroscopies have been used to study the assembly of a hairpin nucleotide sequence (nucleotides 3-30) of the 5' non-coding region of the hepatitis C virus RNA (5'-GGCGGGGAUUAUCCCCGCUGUGAGGCGG-3') with a RNA 20mer ligand (5'-CCGCCUCACAAAGGUGGGGU-3') in the presence of magnesium ion and spermidine. The resulting complex involves two helical structural domains: the first one is an intermolecular duplex stem at the bottom of the target hairpin and the second one is a parallel triplex generated by the intramolecular hairpin duplex and the ligand. Infrared spectroscopy shows that N-type sugars are exclusively present in the complex. This is the first case of formation of a RNA parallel triplex with purine motif and shows that this type of targeting RNA strands to viral RNA duplexes can be used as an alternative to antisense oligonucleotides or ribozymes.

A Single DNA Molecule Nanomotor

Nanomotors consisting of single protein molecules are abundant in living systems. Here we report a nanomotor made of a single DNA molecule. The DNA nanomotor can adopt two distinct conformations, intramolecular tetraplex and intermolecular duplex. The nanomotor switches between the two conformations through alternating DNA hybridization and strand exchange reactions, which enables the nanomotor to perform an inchworm like extending−shrinking motion. When the single molecule nanomotor is loaded with two organic molecules, a fluorophore and a quencher, the motion can be viewed in real time by monitoring the fluorescent signal. The DNA nanomotor functions efficiently both in solution and on nanoparticle surfaces. Its simple yet stable structure, convenient operation, and high efficiency may make the DNA nanomotor practically useful for powering nanosystems in future applications.

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.

Protein-free parallel triple-stranded DNA complex formation.

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

Parallel-Stranded DNA with Mixed AT/GC Composition: Role of trans G·C Base Pairs in Sequence Dependent Helical Stability

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions.

JAZ Requires the Double-stranded RNA-binding Zinc Finger Motifs for Nuclear Localization

We have cloned and characterized a novel zinc finger protein, termed JAZ. JAZ contains four C(2)H(2)-type zinc finger motifs that are connected by long (28-38) amino acid linker sequences. JAZ is expressed in all tissues tested and localizes in the nucleus, primarily the nucleolus. JAZ preferentially binds to double-stranded (ds) RNA or RNA/DNA hybrids rather than DNA. Mutation of individual zinc finger motifs reveals that the zinc finger domains are not only essential for dsRNA binding but are also required for its nucleolar localization, which demonstrates a complex trafficking mechanism dependent on the nucleic acid-binding capability of the protein. Furthermore, forced expression of JAZ potently induces apoptosis in murine fibroblast cells. Thus, JAZ may belong to a class of zinc finger proteins that features dsRNA binding and may regulate cell growth via the unique dsRNA binding properties.

Detection of a novel ATP-dependent cross-linked protein at the 5' splice site-U1 small nuclear RNA duplex by methylene blue-mediated photo-cross-linking.

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5' splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5' splice site. Moreover, although p65 cross-linking requires only a 5' splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

DNA and RNA oligomer sequences from the 3' noncoding region of the chicken glutamine synthetase gene from intramolecular hairpins.

The DNA sequence of the chicken glutamine synthetase gene contains an A.T-rich stretch of approximately 1500 base pairs in the 3' noncoding regions of exon 7 [Pu, H., & Young, A. P. (1989) Gene 18, 169-175]. Within this region several palindromic sequences occur that could conceivably form intramolecular structures. One such perfect inverted repeat sequence resides between positions 2605 and 2623. To investigate the hairpin-forming potential for this sequence, optical and calorimetric melting and gel electrophoresis studies have been performed on the following synthetically prepared DNA and RNA oligomer subsequences: DNA, 5'd-T-T-T-T-T-T-A-A-T-A-A-T-T-A-A-A-A-A-A-3'; and RNA, 5'r-U-U-U-U-U-U-A-A-U-A-A-U-U-A-A-A-A-A-A-3'. The DNA strand corresponds to the coding strand sequence while the RNA strand represents the transcribed mRNA. Results of melting analysis of these 19-base, partially self-complementary strands performed in 115 mM Na+ yielded evaluations of their thermodynamic transition parameters. These values are consistent with the melting of unimolecular structures, presumably hairpins. Thermodynamic parameters evaluated by analysis of the optical melting transitions assuming a two-state model and measured directly by differential scanning calorimetry agreed within experimental error. Therefore, melting behavior of the hairpins is all-or-none like. The DNA hairpin is slightly more stable than the RNA hairpin with melting enthalpy delta H0 = 41.2 +/- 3.8 kcal/mol and entropy delta S0 = 133 +/- 11 cal/K.mol (eu) compared to delta H0 = 32.0 +/- 6.0 kcal/mol and entropy delta S0 = 105 +/- 20 eu for the RNA. Gel electrophoretic analysis of these oligomers alone and in various mixtures with their DNA and RNA complementary strands was also performed. Consistent with interpretations of melting results, these experiments revealed both strands alone preferentially form intramolecular hairpin structures. In mixtures in which their complementary strands are in vast molar excess (stoichiometric ratios > 10:1), the intramolecular structures are converted to intermolecular duplexes. For the DNA and RNA strands examined, the conversion is not complete until over a 1000-fold excess of the complementary strand is added. Semiquantitative analysis of gel electrophoretograms enabled evaluations of the relative free energies of the hairpin and duplex states as a function of complementary strand concentration. With the finding that these sequences preferentially form hairpins, potential roles these structures could play in regulatory activities are considered.

Modified substrates as probes for studying uracil-DNA glycosylase

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(β- d-2′-deoxythreo-pentofuranosil)uracil (xU) or 1-(β- d-3′-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2′-deoxy-2′-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100–200-fold less efficiently than the natural ones. For Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.

Structural Polymorphism of d(GA · TC)n DNA Sequences: Intramolecular and Intermolecular Associations of the Individual Strands

Alternating d(GA · TC)n sequences are highly structurally polymorphic. Most of their conformational flexibility is likely to reside in the structural properties of the individual strands themselves. In this paper the conformational behaviour of the d(GA)20 and d(TC)20 oligonucleotides was analysed. Formation of d(GA)20 intramolecular duplexes is observed at any pH value, from 8·3 to 4·6. On the other hand, intramolecular d(TC)20 duplexes are formed only under acidic conditions. The acid d(TC)20 intramolecular duplex is likely to be stabilized through the formation of C+C pairs, the thymine residues remaining unpaired. The d(GA)20 oligonucleotide also forms intermolecular duplexes which coexist with the intramolecular forms at any pH, from 8·3 to 4·6. The structural conformation adopted by the d(TC)20 oligonucleotide at neutral pH is uncertain. Under these conditions, this oligonucleotide shows an electrophoretic apparent molecular weight consistent with the formation of a bimolecular complex. However, no hydrogen bonding was observed to occur under these conditions. Implications of these results for an understanding of the molecular principles behind the conformational flexibility of alternating d(GA · TC)n sequences are discussed. The possible biological significance of these results is also discussed.

Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA

Antisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity. We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture. We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference. Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation. Inhibition can be substantial (over 70%) but is transient. Transience does not result from mutation of the input virus. Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation. Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs†

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

Cleavage of single stranded oligonucleotides byEcoRI restriction endonuclease

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.

The primary structure of crossover regions of intertypic poliovirus recombinants: A model of recombination between RNA genomes

The nucleotide sequence of crossover sites in the genome of four intertypic (type 3/type 1) poliovirus recombinants has been determined. The approximate boundaries of the crossover regions were first estimated by RNase T1 oligonucleotide mapping of the recombinant genomes; then appropriate regions were sequenced by the chain termination method using oligonucleotide-primed reverse transcription of the recombinant RNAs. The crossover sites (defined as the contiguous sequences shared by the recombinant and both parental genomes flanked, in the recombinant genome, by heterotypic RNA segments) are 5, 5, 7, and 11 nucleotides long, respectively. The recombination was precise and was not accompanied by any other genetic alterations. The recombination sites were found to be located within genome segments having a potential to form secondary structure elements. Based on this observation, a model of recombination between picornaviral RNA genomes has been proposed. The essence of this model consists in bringing together homologous regions of two recombining RNA genomes via formation of intermolecular duplexes, detachment of the nascent 3′ end of the newly synthesized complementary RNA from a “parting” site on the first template and its subsequent “jumping” to the identical (or closely related) “anchoring” site on the other template. Features of this model are discussed in some detail.

Interstrand duplexes in nuclear RNA

AbstractNuclear intermolecular duplexes appear to be a general feature of nucleated cells. Most of these duplexes are formed between large RNA as well as between large and small RNA molecules. A significant portion of the large molecules belong to a special class of RNA that is restricted to the nucleus and, therefore, not designated for export. These molecules are assembled with proteins and form a structure of a higher order. The possibility that these molecules and a set of small nuclear RNAs are components of a complex machine, “the transportosome”, which functions in nucleocytoplasmic traffic, is discussed.

Molecular cloning of the terminal hairpin of vaccinia virus DNA as an imperfect palindrome in an Escherichia coli plasmid

The genome of vaccinia virus is a linear duplex molecule of approximately 185 kb with hairpins at each end that link the complementary strands. The hairpins, which exist in two forms that are inverted and complementary in sequence, were isolated as Xba I restriction fragments and converted to a linear intermolecular duplex structure by denaturation and reannealing. The latter was then stably cloned as a 142-bp imperfect palindrome in an Escherichia coli plasmid. The insert was excised from the plasmid and the palindrome was extended on both sides by ligating it to the adjacent vaccinia virus DNA segment. The resulting fragment was cloned as a 278-bp imperfect palindrome. Restriction endonuclease analysis and DNA sequencing indicated the absence of any deletions or rearrangements. After excision from the plasmid, the palindrome was converted by heating and rapid cooling to the original two hairpin forms. In this manner, large quantities of vaccinia virus telomeres may be obtained for physical and biochemical studies.