Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.

Differential Regulation of Sugar-Sensitive Sucrose Synthases by Hypoxia and Anoxia Indicate Complementary Transcriptional and Posttranscriptional Responses1

The goal of this research was to resolve the hypoxic and anoxic responses of maize (Zea mays) sucrose (Suc) synthases known to differ in their sugar regulation. The two maize Suc synthase genes, Sus1 and Sh1, both respond to sugar and O2, and recent work suggests commonalities between these signaling systems. Maize seedlings (NK508 hybrid, W22 inbred, and an isogenic sh1-null mutant) were exposed to anoxic, hypoxic, and aerobic conditions (0, 3, and 21% O2, respectively), when primary roots had reached approximately 5 cm. One-centimeter tips were excised for analysis during the 48-h treatments. At the mRNA level, Sus1 was rapidly up-regulated by hypoxia (approximately 5-fold in 6 h), whereas anoxia had less effect. In contrast, Sh1 mRNA abundance increased strongly under anoxia (approximately 5-fold in 24 h) and was much less affected by hypoxia. At the enzyme level, total Suc synthase activity rose rapidly under hypoxia but showed little significant change during anoxia. The contributions of SUS1 and SH1 activities to these responses were dissected over time by comparing the sh1-null mutant with the isogenic wild type (Sus+, Sh1+). Sh1-dependent activity contributed most markedly to a rapid protein-level response consistently observed in the first 3 h, and, subsequently, to a long-term change mediated at the level of mRNA accumulation at 48 h. A complementary midterm rise in SUS1 activity varied in duration with genetic background. These data highlight the involvement of distinctly different genes and probable signal mechanisms under hypoxia and anoxia, and together with earlier work, show parallel induction of “feast and famine” Suc synthase genes by hypoxia and anoxia, respectively. In addition, complementary modes of transcriptional and posttranscriptional regulation are implicated by these data, and provide a mechanism for sequential contributions from the Sus1 and Sh1 genes during progressive onset of naturally occurring low-O2 events.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Synthesis and assembly of self-complementary calix[4]arenes.

A calix[4]arene was designed to reversibly dimerize and form an egg-shaped enclosure. Adhesive interactions in the assembly were provided by four self-associating ureas, which form a cyclic array containing 16 hydrogen bonds. The synthesis was completed in four steps from the previously described O,O',O",O"'-tetrabenzylcalix[4]arene. Evidence for dimerization of the calixarene tetraurea was provided by H NMR, mass spectrometry, and the observation of encapsulated molecules. The resulting cavity was of sufficient size to capture guests such as ethyl benzene and p-xylene.

Biologic therapies and non-melanoma skin cancer rates in three chronic immune-mediated diseases

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014