Biomedical subjects

M J Gait

Publications and source records attributed to M J Gait.

At least 19 recordsLinked to original sources

PubMed2007

Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides.

CPPs (cell-penetrating peptides) have given rise to much interest for the delivery of biomolecules such as peptides, proteins or ONs (oligonucleotides). CPPs and their conjugates were initially thought to translocate through the cell membrane by a non-endocytotic mechanism which has recently been re-evaluated. Basic-amino-acid-rich CPPs first interact with cell-surface proteoglycans before being internalized by endocytosis. Sequestration and degradation in endocytotic vesicles severely limits the cytoplasmic and nuclear delivery of the conjugated biomolecules. Accordingly, splicing correction by CPP-conjugated steric-block ON analogues is inefficient in the absence of endosomolytic agents. New arginine-rich CPPs allowing efficient splicing correction by conjugated PNAs (peptide nucleic acids) or PMO (phosphorodiamidate morpholino oligomer) steric blockers in the absence of endosomolytic agents have recently been defined in our group and are currently being characterized. They offer promising leads for the development of efficient cellular delivery vectors for therapeutic steric-block ON analogues.

Alternative Splicing↗

PubMed2005

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.

Animals↗

PubMed2001

Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2'-O-methyl/LNA oligoribonucleotides.

The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2'-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7xOMe and 5x5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100-200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3'-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3'-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3'-FAM 12-residue 7xOMe/5xLNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3'-FAM OMe 12-mer. An alternating PS-containing 3'-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.

Cations↗

PubMed2001

Novel spermine-amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration.

Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of L-diaminobutyric acid (Dab). Of a range of novel synthetic spermine-amino acid conjugates, L-Dab-spermine (but not D-Dab nor other amino acid conjugates) was more effective than spermine itself.

Amino Acids↗

PubMed2001

Synthesis of peptide-oligonucleotide conjugates: application to basic peptides.

Efficient methods of synthesis of peptide-oligonucleotide conjugates are badly needed for studies of uptake into cells in culture. We describe improvements to the procedures involved in "native ligation" conjugation in solution to extend the method to basic peptides. Further, we describe progress in development of a total solid-phase synthesis approach that makes use of a L-homoserine linker as the key reagent for growing of oligonucleotide and peptide chains on a single solid support.

Amino Acid Sequence↗

PubMed2001

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Acetylcholinesterase↗

PubMed2000

Efficient conjugation of peptides to oligonucleotides by "native ligation".

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.

Amino Acid Sequence↗

PubMed2000

Investigation of the proposed interdomain ribose zipper in hairpin ribozyme cleavage using 2'-modified nucleosides.

The hairpin ribozyme achieves catalytic cleavage through interaction of essential nucleotides located in two distinct helical domains that include internal loops. Initial docking of the two domains is ion dependent and appears to be followed by a structural rearrangement that allows the ribozyme to achieve a catalytically active state that can undergo cleavage. The proposed structural rearrangement may also be ion dependent and is now of increased importance due to recent evidence that docking is not rate limiting and that metal ions are unlikely to be involved in the chemical cleavage step. An initial structural model of the docked hairpin ribozyme included a proposal for a ribose zipper motif that involves two pairs of hydroxyl groups at A(10) and G(11) in domain A pairing with C(25) and A(24) in domain B, respectively. We have used a chemical functional group substitution technique to study whether this proposed ribose zipper is likely to be present in the active, conformationally rearranged ribozyme that is fit for cleavage. We have chemically synthesized a series of individually modified hairpin ribozymes containing 2'-analogues of nucleosides, that include 2'-deoxy and 2'-deoxy-2'-fluoro at each of the four nucleoside positions, 2'-amino-2'-deoxy, 2'-deoxy-2'-thio, and 2'-arabino at position C(25), and 2'-oxyamino at position A(10), as well as some double substitutions, and we studied their cleavage rates under both single- and multiple-turnover conditions. We conclude that at least some of the hydrogen-bonding interactions in the ribose zipper motif, either as originally proposed or in a recently suggested structural variation, are unlikely to be present in the active rearranged form of the ribozyme that undergoes cleavage. Instead, we provide strong evidence for a very precise conformational positioning for the residue C(25) in the active hairpin. A precise conformational requirement would be expected for C(25) if it rearranges to form a base-triple with A(9) and the essential residue neighboring the cleavage site G(+1), as recently proposed by another laboratory. Our results provide further support for conformational rearrangement as an important step in hairpin ribozyme cleavage.

Base Sequence↗

PubMed2000

Fibroblast growth factor (FGF) receptor 1-IIIb is a naturally occurring functional receptor for FGFs that is preferentially expressed in the skin and the brain.

Fibroblast growth factors (FGFs) transmit their signals through four transmembrane receptors that are designated FGFR1-4. Alternative splicing in the extracellular region of FGFR1-3 generates receptor variants with different ligand binding affinities. Thus two types of transmembrane receptors (IIIb and IIIc isoforms) have been identified for FGFR2 and FGFR3, and the existence of analogous variants has been postulated for FGFR1 based on its genomic structure. However, only a single full-length transmembrane FGFR1 variant (FGFR1-IIIc) has been identified so far. Here we describe the cloning of a full-length cDNA encoding FGFR1-IIIb from a mouse skin wound cDNA library. This receptor isoform was expressed at the highest levels in a subset of sebaceous glands of the skin and in neurons of the hippocampus and the cerebellum. FGFR1-IIIb was expressed in L6 rat skeletal muscle myoblasts and used in cross-linking and receptor binding studies. FGF-1 was found to bind the receptor with high affinity, whereas FGF-2, -10, and -7 bound with significantly lower affinities. Despite their apparently similar but low affinities, FGF-10 but not FGF-7 induced the activation of p44/42 mitogen-activated protein kinase in FGFR1-IIIb-expressing L6 myoblasts and stimulated mitogenesis in these cells, demonstrating that this new receptor variant is a functional transmembrane receptor for FGF-10.

Amino Acid Sequence↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

The harpin ribozyme: the roles of ions in RNA cleavage.

The cleavage reaction of the hairpin ribozyme is facilitated by divalent metal ions, such as Mg2+, or by non-metallic polycations, such as the polyamine spermine. We show substantial enhancement of cleavage with combinations of metallic and non-metallic ions. In addition we elucidate the locations of some ion binding sites by Fenton chemistry.

Base Sequence↗

PubMed1998

Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells.

Aminoglycosides↗

PubMed1998

Site-specific cross-linking of amino acids in the basic region of human immunodeficiency virus type 1 Tat peptide to chemically modified TAR RNA duplexes.

The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem-loop known as the trans-activation responsive region (TAR) that occurs in all viral transcripts. We have photochemically cross-linked to Tat peptide (37-72), a model TAR duplex substituted at U23 in the bulge by 4-thioU. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI-TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly 13C and 15N isotopically labeled arginine. We also describe a new chemical procedure for obtaining site-specific cross-links to Tat via the use of 2'-beta-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U23-2'-functionalized TAR was shown to cross-link uniquely to Lys51 in the basic region of Tat, whereas other sites in the upper and lower stems of TAR (U35, U38, and U42) showed cross-linking only to the N-terminus of Tat peptide (37-72). U40 cross-linked to both Lys51 and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of Tat peptide to the major groove of TAR RNA.

Affinity Labels↗

PubMed1998

RNA recognition and regulation of HIV-1 gene expression by viral factor Tat.

Viral transcription factor Tat is a small nuclear protein containing a large number of basic amino acids. The tat gene consists of two exons but only the first encoding 72-amino acid polypeptide is necessary for protein activity. Since the second exon is poorly conservative the total number of amino acids among Tat proteins from different strains of HIV-1 varies from 86 to 130. Tat protein acts as trans-activator of HIV genome transcription. It is absolutely required for viral functioning. Tat increases processivity of RNA-polymerase II by abolition of transcription blockade, which appears after polycondensation of the first 60-70 nucleotides of either HIV mRNA, i.e., it acts as antiterminator. For manifestation of its activity Tat specifically binds to the double stranded RNA fragment called TAR which is located at the 5'-terminus of all HIV mRNAs. The TAR structure contains a hairpin and a side loop. The Tat-binding region includes only a site of the loop; manifestation of Tat activity in vivo requires the full TAR and additional cellular co-factors.

Amino Acid Sequence↗

PubMed1997

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.

Computer Simulation↗

PubMed1997

Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues.

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.

Amino Acid Sequence↗

PubMed1997

Progress toward the structure and therapeutic use of the hairpin ribozyme.

The hairpin ribozyme is one of a number of small catalytic RNAs that are excellent paradigms for RNA structure-function analysis and have potential also as therapeutic agents. This review outlines current understanding of the structure of the hairpin ribozyme and its basis for catalytic activity. Included also is a discussion of the functional group requirements for cleavage and the first steps being taken to understanding its folding. Finally, recent developments are highlighted in engineering the hairpin ribozyme for intracellular use as a potential gene therapy agent.

Base Composition↗

PubMed1996

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.

Animals↗