Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Structure-activity relationships of aminoglycoside-arginine conjugates that bind HIV-1 RNAs as determined by fluorescence and NMR spectroscopy

We present here a new set of aminoglycoside-arginine conjugates (AACs) that are either site-specific or per-arginine conjugates of paromomycin, neamine, and neomycin B as well as their structure-activity relationships. Their binding constants (KD) for TAR and RRE RNAs, measured by fluorescence anisotropy, revealed dependence on the number and location of arginines in the different aminoglycoside conjugates. The binding affinity of the per-arginine aminoglycosides to TAR is higher than to RRE, and hexa-arginine neomycin B is the most potent binder (KD=5 and 23 nM, respectively). The 2D TOCSY NMR spectrum of the TAR monoarginine-neomycin complex reveals binding at the bulge region of TAR.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B

Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts.     

Aminoglycoside binding to human and bacterial A-Site rRNA decoding region constructs

The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.     

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.     

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.     

Stereochemical control of small molecule binding to bulged DNA: comparison of structures of spirocyclic enantiomer-bulged DNA complexes

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and ent-DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. Ent-DDI is a left-handed wedge-shaped spirocyclic molecule whose aglycone portion is an enantiomer of DDI, which mimics the spirocyclic geometry of the natural product, NCSi-gb, formed by base-catalyzed activation of the enediyne antibiotic neocarzinostatin. The benzindanone moiety of ent-DDI intercalates between the A6.T21 and the T9.A20 base pairs, overlapping with portions of the purine bases; the dihydronaphthalenone moiety is positioned in the minor groove along the G7-T8-T9 bulge sequence; and the aminoglycoside is in the middle of the minor groove, approaching A20 of the nonbulged strand. This alignment of ent-DDI along the DNA helical duplex is in the reverse direction to that of DDI. The aminoglycoside moiety of ent-DDI is positioned in the 3' direction from the bulge region, whereas that of the DDI is positioned in the 5' direction from the same site. This reverse binding orientation within the bulge site is the natural consequence of the opposite handedness imposed by the spirocyclic ring junction and permits the aromatic ring systems of the two spirocyclic enantiomers access to the bulge region. NMR and CD data indicate that the DNA in the DDI-bulged DNA complex undergoes a larger conformational change upon complex formation in comparison to the ent-DDI-bulged DNA, explaining the different binding affinities of the two drugs to the bulged DNA. In addition, there are different placements of the bulge bases in the helical duplex in the two complexes. One bulge base (G7) stacks inside the helix, and the other one (T8) is extrahelical in the DDI-bulged DNA complex, whereas both bulge bases in the ent-DDI-bulged DNA complex prefer extrahelical positions for drug binding. Elucidation of the detailed binding characteristics of the synthetic spirocyclic enantiomers provides a rational basis for the design of stereochemically controlled drugs for bulge binding sites.     

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.     

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.     

Solution structure of a wedge-shaped synthetic molecule at a two-base bulge site in DNA

The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes.     

Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.     

Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer.

BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.     

Conformational transitions in RNA single uridine and adenosine bulge structures: a molecular dynamics free energy simulation study

Extra unmatched nucleotides (single base bulges) are common structural motifs in folded RNA molecules and can participate in RNA-ligand binding and RNA tertiary structure formation. Often these processes are associated with conformational transitions in the bulge region such as flipping out of the bulge base from an intrahelical stacked toward a looped out state. Knowledge of the flexibility of bulge structures and energetics of conformational transitions is an important prerequisite to better understand the function of this RNA motif. Molecular dynamics simulations were performed on single uridine and adenosine bulge nucleotides at the center of eight basepair RNA molecules and indicated larger flexibility of the bulge bases compared to basepaired regions. The umbrella sampling method was applied to study the bulge base looping out process and accompanying conformational and free energy changes. Looping out toward the major groove resulted in partial disruption of adjacent basepairs and was found to be less favorable compared to looping out toward the minor groove. For both uridine and adenosine bulges, a positive free energy change for full looping out was obtained which was approximately 1.5 kcal mol-1 higher in the case of the adenosine compared to the uridine bulge system. The simulations also indicated stable partially looped out states with the bulge bases located in the RNA minor groove and forming base triples with 5'-neighboring basepairs. In the case of the uridine bulge this state was more stable than the intrahelical stacked bulge structure. Induced looping out toward the minor groove involved crossing of an energy barrier of approximately 3.5 kcal mol-1 before reaching the base triple state. A continuum solvent analysis of intermediate bulge states indicated that electrostatic interactions stabilize looped out and base triple states, whereas van der Waals interactions and nonpolar contributions favor the stacked bulge conformation.     

RNA recognition by Tat-derived peptides: interaction in the major groove?

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.     

Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex

Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions. Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe). Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement. The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions. Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase. Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E. coli tRNA(Phe) in the mid microM range. Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.     

Towards artificial ribonucleases: the sequence-specific cleavage of RNA in a duplex.

Lanthanide complexes covalently attached to oligonucleotides have been shown to cleave RNA in a sequence-specific manner. Efficient cleavage, however, is at present limited to single-stranded RNA regions, as RNA in a duplex is considerably more resistant to strand scission. To overcome this limitation, we have designed and synthesised artificial nucleases comprising lanthanide complexes covalently linked to oligodeoxyribonucleotides which cleave a partially complementary RNA at a bulged site, in the duplex region. Strand scission occurs at or near the bulge. Cleavage of the RNA target by the metal complex can be addressed via the major or the minor groove. In an example of a competitive situation, where the cleavage moiety has access to both a bulge and a single-strand region, transesterification at the bulge is favoured. Such artificial ribonucleases may find application as antisense agents and as tools in molecular biology. In addition, the results may have importance for the design of artificial ribonucleases which are able to act with catalytic turnover.     

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.