High-throughput discovery of organic cages and catenanes using computational screening fused with robotic synthesis.

Supramolecular synthesis is a powerful strategy for assembling complex molecules, but to do this by targeted design is challenging. This is because multicomponent assembly reactions have the potential to form a wide variety of products. High-throughput screening can explore a broad synthetic space, but this is inefficient and inelegant when applied blindly. Here we fuse computation with robotic synthesis to create a hybrid discovery workflow for discovering new organic cage molecules, and by extension, other supramolecular systems. A total of 78 precursor combinations were investigated by computation and experiment, leading to 33 cages that were formed cleanly in one-pot syntheses. Comparison of calculations with experimental outcomes across this broad library shows that computation has the power to focus experiments, for example by identifying linkers that are less likely to be reliable for cage formation. Screening also led to the unplanned discovery of a new cage topology-doubly bridged, triply interlocked cage catenanes.

Reticular synthesis of porous molecular 1D nanotubes and 3D networks.

Synthetic control over pore size and pore connectivity is the crowning achievement for porous metal-organic frameworks (MOFs). The same level of control has not been achieved for molecular crystals, which are not defined by strong, directional intermolecular coordination bonds. Hence, molecular crystallization is inherently less controllable than framework crystallization, and there are fewer examples of 'reticular synthesis', in which multiple building blocks can be assembled according to a common assembly motif. Here we apply a chiral recognition strategy to a new family of tubular covalent cages to create both 1D porous nanotubes and 3D diamondoid pillared porous networks. The diamondoid networks are analogous to MOFs prepared from tetrahedral metal nodes and linear ditopic organic linkers. The crystal structures can be rationalized by computational lattice-energy searches, which provide an in silico screening method to evaluate candidate molecular building blocks. These results are a blueprint for applying the 'node and strut' principles of reticular synthesis to molecular crystals.

Effect of gallium on growth of Streptococcus mutans NCTC 10449 and dental tissues.

Gallium-doped phosphate-based glasses (Ga-PBG) were assessed for their impact on Streptococcus mutans and dental mineralisation, firstly by disc diffusion assays followed by biofilms grown on nitrocellulose filter membrane (NFM) and constant-depth film fermentor (CDFF). Short-time exposure (10 min) effects of Ga-PBG on S. mutans biofilm were compared with that of 0.2% chlorhexidine. The effects of Ga-PBG on bovine enamel (which was investigated under pH-cycling condition) and dentine were analysed using transverse microradiography (TMR), profilometry and inductively coupled plasma optical-emission spectrometry (ICP-OES). The disc diffusion assays showed inhibition zones of 24.5 ± 0.5 mm for Ga-PBG compared with controls (C-PBG). Ga-PBG showed statistically significant growth inhibition of S. mutans biofilms on NFM (p = 0.001) and CDFF (p < 0.046) compared with hydroxyapatite (HA) and C-PBG. The CDFF assay revealed a maximum of 2.11 log colony-forming unit (CFU) reduction at 48 h, but short-time exposure effects were comparable with that of 0.2% chlorhexidine only on older biofilms (maximum of 0.59 vs. 0.69 log CFU reduction at 120 h). TMR analyses of the enamel revealed non-significant mineral loss (p = 0.37) only in the case of Ga-PBG samples compared with controls including sodium fluoride. ICP-OES analyses indicated transient gallium adsorption into dentine by calcium displacement. The results confirmed that gallium inhibited S. mutans growth and appears to have the potential to protect the enamel surface under conditions representative of the oral environment. Further work is needed to establish whether it has an application in daily oral hygiene procedures to prevent or reduce caries.

Improved methods for structural studies of proteins using nuclear magnetic resonance spectroscopy.

The past few years have seen the development of three- and four-dimensional heteronuclear nuclear magnetic resonance methods. Increased sophistication in labelling strategies, use of pulse-field gradients and the application of these methods at higher magnetic fields has, in combination with improved software, allowed studies of the structure, interactions and dynamics of significantly larger proteins (now up to approximately 270 amino acid residues).

Minimisation of sensitivity losses due to the use of gradient pulses in triple-resonance NMR of proteins.

The use of pulsed field gradients in multiple-pulse NMR experiments has many advantages, including the possibility of obtaining excellent water suppression without the need for selective presaturation. In such gradient experiments the water magnetization is dephased deliberately; exchange between the saturated protons of the solvent water and the NH protons of a protein transfers this saturation to the protein. As the solvent is in large excess and relaxes relatively slowly, the result is a reduction in the sensitivity of the experiment due to the fact that the NH proton magnetization is only partially recovered. These effects can be avoided by ensuring that the water magnetization remains intact and is returned to the +z-axis at the start of data acquisition. General procedures for achieving this aim in any triple-resonance experiment are outlined and two specific examples are given. Experimental results confirm the sensitivity advantage of the modified sequences.

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding.

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.

Localization of the control region for expression of exotoxin A in Pseudomonas aeruginosa.

The 2,760-base-pair (bp) PstI-EcoRI segment of the chromosome of Pseudomonas aeruginosa PA103 which carries the exotoxin A structural gene was expressed from an internal promoter when cloned in a pUC9 derivative and transformed into a nontoxigenic mutant of P. aeruginosa PAO1. The unique terminal EcoRI site was deleted, and a new EcoRI site was substituted for a PvuI site located 107 bp 5' to the transcription initiation site. Following EcoRI cleavage, Bal31 deletions were generated from this site, and an EcoRI linker sequence, GGAATTCC, was inserted in place of the deleted DNA. Mutants with deletions located 73 bp or more upstream of the transcription initiation site retained normal expression, whereas in mutants with deletions extending into the region 69 bp or less upstream of this site, exotoxin synthesis was greatly reduced. From a KpnI site located 473 bp 3' to the transcription initiation site, a similar series of Bal31 deletion mutants were generated in which the inserted EcoRI linker sequence was located within the same 72-bp region. Pairs of mutants from the two deletion series were identified in which the EcoRI linker was located at the same sequence, and these mutant pairs were ligated to derive a series of constructs in which the EcoRI linker sequence GGAATTCC was substituted for an 8-bp sequence within the 72-bp region. Some of these linker-substituted mutants showed greatly reduced exotoxin A synthesis. The results are consistent with a binding site for a positive activator contiguous with the binding site for an RNA polymerase.

Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa.

The exotoxin A genes from Pseudomonas aeruginosa strains PA103 and PAO1 have been independently cloned in a pUC9-derived plasmid. In a non-toxigenic mutant of PAO1 as host, the cloned genes directed the synthesis of intact exotoxin A that expressed ADP-ribosyltransferase activity upon treatment with urea and dithiothreitol. Western-blot analysis of culture supernatants identified a polypeptide of 67 kDa, the molecular mass of intact exotoxin A. There was an approximately 15-fold increase in the toxin yield from P. aeruginosa cells carrying a cloned PA103 gene compared to PA103, and a 40-fold increase in the yield of toxin gene yielded about four times more toxin than those carrying the cloned PAO1 gene. Toxin expression was correlated with the presence of a transcript that was initiated 88 bp upstream from the translational start site. Little or no messenger RNA from either cloned gene could be detected in an Escherichia coli host, or in a P. aeruginosa host grown in the presence of 0.1 mM-Fe2+, a condition that inhibits toxin expression. The nucleotide sequences of two regions, each of approximately 500 bp, near the 5' and 3' termini of the structural gene were established. In these regions, three exotoxin A gene from PAO1 has ten base-pair differences compared to the PA103 gene, three in the non-coding region, and seven in the structural gene, four of which should lead to amino-acid differences. No apparent sequence similarities were found between the inferred promoter region of the exotoxin A gene and that of other Pseudomonas genes, nor with the consensus sequence of E. coli promoters.

Nucleotide sequence comparisons of plasmids pHD131, pJB1, pFA3, and pFA7 and beta-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae.

The sites of initiation for beta-lactamase mRNA transcription and the nucleotide sequences of beta-lactamase plasmids derived from Haemophilus and Neisseria species were determined. In N. gonorrhoeae, transcription from plasmid pFA3 was initiated from two sites, one located about 20 base pairs (bp) and the other 210 bp upstream of the beta-lactamase initiating codon, whereas in H. influenzae, transcriptional initiation from plasmid pHD131 occurred at two different sites, approximately 150 and 170 bp upstream of the initiating codon. When these plasmids were transformed into Escherichia coli, transcription was initiated at the 150- and 170-bp upstream sites in both plasmids. The nucleotide sequences of both plasmids within the noncoding region upstream of the transcriptional initiation site were identical and, except at two or three nucleotide positions, the sequences were also identical to the corresponding region of Tn3. At one of these positions there is a TA for CG substitution, which correlates in E. coli and Haemophilus sp. with the presence of two strong, overlapping beta-lactamase promoters, initiating transcription at the 150- and 170-bp upstream sites. Over a larger (875-bp) segment comprising most of the sequences of the tnpR and bla genes, the nucleotide sequences of both plasmids were also identical, and although this sequence differed from the corresponding Tn3 sequence at 18 sites, it was identical to that of Tn2, except at one site. The sequence of a second Haemophilus plasmid, pJB1, was identical to that of pHD131 in the same region, except at two nucleotides. All three plasmids were identical in nucleotide sequence in other TnA regions, as well as in regions flanking the TnA sequence, except that the Neisseria plasmid lacked a TnA segment of 3,298 bp [comprising the IR(L) and proximal sequences] together with approximately 273 bp of the non-TnA region adjacent to IR(L). The sequence of a second N. gonorrhoeae plasmid, pFA7, was identical to pFA3, except that the terminal, 3,299 TnA nucleotides were missing.

Variations between the nucleotide sequences of Tn1, Tn2, and Tn3 and expression of beta-lactamase in Pseudomonas aeruginosa and Escherichia coli.

Using S1 nuclease assays, we located the sites of initiation of transcription of the beta-lactamase gene on Tn1 and Tn2. Transcription in Tn2, like that in Tn3, occurred from the P3 promoter, whereas transcription in Tn1 was initiated by two stronger and overlapping promoters, Pa and Pb. The nucleotide sequences of Tn1 and Tn2 were determined over a 1,195-base-pair segment constituting most of the sequences of the tnpR and bla genes and the intervening region. There were six base-pair differences between Tn1 and Tn3. One in the bla regulatory region accounted for the presence of the Pa and Pb promoters, and another in the bla structural gene is consistent with the isoelectric focusing difference found between the Tn1 and Tn3 enzymes. In contrast, there were 24 base-pair differences between Tn2 and Tn3, most of them clustered in one segment of the tnpR gene.

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.

Absence of direct repeats flanking transposons resulting from intramolecular transposition.

Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3. The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660. In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation. In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.

Two improved promoter sequences for the beta-lactamase expression arising from a single base-pair substitution.

A mutation in the transposon Tn2660 derived from the plasmid R6K, and resulting in an approximate tenfold increase in penicillin resistance is shown to be a single GC to AT substitution 177 base pairs 'upstream' of the initiation codon of the structural beta-lactamase (bla) gene. This substitution leads to the transcription of two new mRNAs which can be ascribed to the creation of two new overlapping promoter sequences. All the sequences (450bp) examined in the wild-type Tn2660 are identical to those reported in Tn3.

Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats.

Inverse repeats of the transposon Tn2660 in either a ColE1 or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (less than 2%) evidence of recombination between the repeats after 60 generations of growth in either recA or RecA+ hosts. In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in the recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of the transient intermediate structure. It is concluded that in recA or recA+ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed. A model to explain this difference depends upon a mechanisms that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon.

Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660.

Intermolecular transposition of Tn2660 into pCR1 was measured at 30 degrees C in recA- and recA+ hosts as between 2.6 and 5.5 X 10(-3), a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10(4) transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10(-3) and 10(-4) when a mutant Tn2660 (resulting in the synthesis of a temperature-sensitive beta-lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.