Cell penetrating peptides: overview and applications to the delivery of oligonucleotides.

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.

RNA recognition by the human immunodeficiency virus Tat and Rev proteins.

The human immunodeficiency virus (HIV-1) regulatory proteins, Tat and Rev, are important potential targets for the development of new drug therapies against HIV infection. Both proteins are highly specific RNA-binding proteins that recognize cis-acting regulatory elements in the viral mRNAs. These interactions are fascinating paradigms of a new principle of RNA recognition in which the protein makes contact with functional groups displayed in a distorted major groove of an RNA duplex.

Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA.

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.

Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents.

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription.

The effects of mutations in human immunodeficiency virus type-1 (HIV-1) long terminal repeat on initiation and on Tat-mediated trans-activation were studied using cell-free transcription assays. All the elements that are necessary for efficient transcription initiation in vitro are included in the core promoter. This region contains three tandem Sp1 binding sites, a TATA element and an initiator (INR) sequence. Although the HIV-1 INR element overlaps the trans-activation response region (TAR), it forms an integral part of the promoter. The HIV-1 INR element was characterised in detail using a template that carries a complete HIV-1 promoter and a displaced TAR RNA element. The results demonstrate that the sequence G+1GGTCT is essential for HIV-1 INR function. RNase protection experiments show that Tat acts exclusively to stimulate transcriptional elongation. Mutations in the core promoter elements reduce initiation rates dramatically but do not block Tat activity. For each mutation studied, the total level of transcription in the presence of Tat is proportional to the rate of initiation in the absence of Tat. Furthermore the rate of initiation remains constant in the presence or absence of Tat. We conclude that the elements of the HIV-1 core promoter act in concert to simulate initiation. By contrast, Tat acts independently of the core promoter elements and stimulates elongation. The data strongly suggest that Tat is recruited to the elongating transcription complex during its transit through TAR.

Interactions of INS (CRS) elements and the splicing machinery regulate the production of Rev-responsive mRNAs.

The human immunodeficiency virus type (HIV-1) Rev protein stimulates the export to the cytoplasm of unspliced HIV-1 mRNAs carrying the Rev response element (RRE). However, simple addition of the RRE to beta-globin pre-mRNA does not confer a Rev response on this heterologous transcript. In this paper, we demonstrate that a strong Rev response is conferred on beta-globin pre-mRNA when an inhibitory (INS) element is inserted into the gene together with the RRE. In the presence of INS element, Rev was able to stimulate the export to the cytoplasm of unspliced mRNA 10 to 15-fold. INS elements from the HIV-1 p17 gag and pol genes were equally active in complementing Rev-dependent nuclear export of unspliced mRNA. By contrast, mutated p17 gag INS element, known to be inactive in gag mRNA instability assays, was unable to complement the Rev/RRE system and stimulate nuclear export. Similarly, AUUUA-instability elements from the granulocyte-macrophage colony stimulating factor mRNA (GM-CSF) destabilised beta-globin mRNA but could not substitute for the HIV INS elements. Complementation between the Rev/RRE system and the INS elements was only observed when splicing was efficient. When splicing of the beta-globin gene receptor is impaired by mutations in the 5' splice donor, the 3' splice acceptor sequence, or the polypyrimidine tract, the majority of the unspliced mRNA is exported from the nucleus in the absence of Rev. In the presence of splice site mutations, Rev is able to act independently of a functional INS element and increase the export of unspliced mRNA three to fivefold. We propose that nuclear factor(s) binding to INS elements separate unspliced beta-globin pre-mRNA from the splicing apparatus. Pre-mRNA in this "INS compartment" remains accessible to Rev. Thus, there is a synergy between the INS elements and Rev which leads to enhanced nuclear export of unspliced mRNA.

Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene.

Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains. In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions. Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction. stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined. Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs). We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure.

To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.

Hydrogen-bonding contacts in the major groove are required for human immunodeficiency virus type-1 tat protein recognition of TAR RNA.

The binding site for tat on TAR RNA was analysed by preparing a series of model RNA substrates carrying site-specific functional group modifications. The test RNAs were prepared by annealing two short synthetic oligoribonucleotides to form a duplex structure with a U-rich bulge and flanking sequences identical to TAR RNA. Tat binds these duplex RNAs with approximately half the affinity for wild-type TAR RNA. Substitution at positions U23 or U25 by the base analogue, O4-methyl-dT, which is deficient in its ability to hydrogen-bond at the N3 position reduces tat affinity more than 20-fold. Modifications to purines in the stem of TAR RNA that affect hydrogen-bonding ability in either the major or the minor groove of duplex RNA were also tested. Removal of the nitrogen atom at either the N7 position of G26 or at the N7 position of A27 reduces tat affinity 10- to 20-fold. By contrast removal of the exocyclic amino group in the minor groove at position G26, by substitution with inosine, does not affect tat binding significantly. A single methylphosphonate substitution at the phosphate bond between A22 and U23 also leads to a significant loss of tat binding ability, whereas all other methylphosphonate substitutions in the U-rich bulge are not harmful to tat binding. We conclude that tat forms multiple specific hydrogen bonds to a series of dispersed sites displayed in the major groove of the TAR RNA molecule. These include the N3-H of U23, the N7 of G26, the N7 of A26 and the phosphate between A22 and U23.

High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA/RNA synthesis and labelling.

Reliable methods of machine-aided RNA synthesis have been established to complement those for DNA assembly. Oligonucleotides containing thio-modified backbones and 2'-O-alkyl sugars head the list of many newly available analogues. Biotin, fluorescent agents and many reporter groups can be conveniently introduced into oligonucleotides in multiples by phosphoramidite or H-phosphonate chemistry.

Progress in anti-HIV structure-based drug design.

The course of drug development for the treatment of HIV-1 infection and AIDS is being revolutionized by high-resolution structures of essential viral proteins. We survey the impact on drug design of the recently elucidated structural knowledge of two essential enzymes, reverse transcriptase and protease, and three new targets, the viral integrase and the gene regulatory protein-RNA interactions, Tat-TAR and Rev-RRE.

A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI.

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.

Synthetic oligoribonucleotides carrying site-specific modifications for RNA structure-function analysis.

Synthetic oligoribonucleotides have become increasingly valuable in studies of RNA structure and function. A range of nucleotide analogues is available which carry modifications in the base, sugar or phosphate moieties. Such analogues have been incorporated into synthetic RNA structures to eliminate or alter individual functional groups in the RNA which potentially can take part in hydrogen-bonding or other non-covalent interactions. Comparisons of the properties of the modified RNAs with unmodified RNA models allow conclusions to be drawn concerning the importance or otherwise of specific functional groups within the RNA. These methods have been applied to studies of RNA interactions with proteins, RNA catalysis and RNA structure.

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous Visualization of Vasopressin and Oxytocin mRNA- Containing Neurons in the Hypothalamus Using Non-Radioactive in situ Hybridization Histochemistry.

In situ hybridization histochemistry (ISHH) using synthetic oligodeoxynucleotide probes has been used to demonstrate the sites of expression of mRNA for vasopressin (AVP) and oxytocin (OXT) in the rat brain. ISHH was performed with two types of non-radioactive probes, labelled with either alkaline phosphatase or 5'-biotin. Simultaneous detection of the mRNAs for both AVP and OXT was achieved using an alkaline phosphatase substrate for the AVP probe and an anti-biotin monoclonal (mouse) antibody for the OXT probe. These probes revealed two non-overlapping populations of AVP and OXT neurons on the same section.

In situ hybridization histochemistry: localisation of vasopressin mRNA in rat brain using a biotinylated oligonucleotide probe.

A biotinylated antisense oligonucleotide probe specific for the glycopeptide sequence of arginine vasopressin mRNA has been used with amplified detection for visualisation of arginine vasopressin mRNA in the rat hypothalamus. RNAase pretreatment to destroy arginine vasopressin mRNA and use of excess complementary oligonucleotide (sense) to absorb the biotinylated antisense oligonucleotide demonstrated the reaction is specific for arginine vasopressin mRNA. Further, dehydration of rats using 2% saline resulted in an increase in specific staining. The staining is localized to those neurones in the hypothalamus known to contain arginine vasopressin.

New phosphoramidite reagents for the synthesis of oligonucleotides containing a cysteine residue useful in peptide conjugation.

The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-alpha-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5'-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via "native ligation".