title: Enhanced targeting of triplex forming oligonucleotides

authors: Peters, Linda M.

abstract: Oligonucleotides offer the potential to inhibit the expression of specific genes within living cells by binding to the major groove of the DNA helix in a sequence-specific manner to form a DNA triple helix. The ultimate application of such oligonucleotides is to act as selective therapeutic tools to suppress transcription, inhibit replication and induce site specific mutations in mammalian genes. However, to date, most triplex experiments have utilised simple in vitro systems and it has proved rather more difficult to demonstrate selective actions in vivo.

In this study, a series of psoralen-conjugated oligonucleotides (10-17 nt) were designed to target specific bovine repetitive sequences in the male Y chromosome. Repetitive Y chromosome sequences described by Reed et al., 1995 and Grobet et al., 1996 were investigated for efficient DNA triplex formation. The rationale in this approach was that the presence of several hundred copies rather than just one target per genome would greatly enhance the efficiency of DNA triplex formation in vivo. Psoralen-conjugated oligonucleotides designed to these sequences were screened by performing DNA triplex binding assays and analysing photoadduct formation by electrophoretic mobility shift assays (EMSA).

Six psoralen-conjugated oligonucleotides were found to selectively and efficiently intercalate to their bovine dsDNA target sequences (38-49 bp) in vitro after irradiation with UV light (360 nm), forming covalent DNA photoadducts. Stable photoadducts were also detected when these oligonucleotides were targeted to larger dsDNA targets, ranging from 0.1 to 3 Kb in size. In general, a small UV dose (0.03 J/cm²) was sufficient to form monoadducts, with crosslinks forming after fifteen minutes of UV irradiation (1 J/cm²). The efficiency of bovine monoadduct formation was similar to that in the well-publicised supF DNA triplex system.

PCR and Southern blot analyses also showed that the repetitive Y-chromosomal DNA region (100-400 bp) was indeed male specific and present in multiple copies. However, when Southern blot analyses were carried out using oligonucleotide probes (16-49 nt) which corresponded to the triplex target sequence within this region, relatively weak hybridisation was observed, suggesting that these specific sequences were not present in such high copies. Although this data indicates that the male specific repeat regions must contain segments of consensual rather than exact sequences, high quality non-degenerate DNA sequencing data in the triplex targeting region was obtained from the male specific PCR products. Clearly, the exact nature of these male specific repeats requires further investigation to establish the effects of targeting psoralen-conjugated oligonucleotides to male genomic DNA as opposed to female genomic DNA.

text: Enhanced targeting of triplex forming oligonucleotides Oligonucleotides offer the potential to inhibit the expression of specific genes within living cells by binding to the major groove of the DNA helix in a sequence-specific manner to form a DNA triple helix. The ultimate application of such oligonucleotides is to act as selective therapeutic tools to suppress transcription, inhibit replication and induce site specific mutations in mammalian genes. However, to date, most triplex experiments have utilised simple in vitro systems and it has proved rather more difficult to demonstrate selective actions in vivo.

In this study, a series of psoralen-conjugated oligonucleotides (10-17 nt) were designed to target specific bovine repetitive sequences in the male Y chromosome. Repetitive Y chromosome sequences described by Reed et al., 1995 and Grobet et al., 1996 were investigated for efficient DNA triplex formation. The rationale in this approach was that the presence of several hundred copies rather than just one target per genome would greatly enhance the efficiency of DNA triplex formation in vivo. Psoralen-conjugated oligonucleotides designed to these sequences were screened by performing DNA triplex binding assays and analysing photoadduct formation by electrophoretic mobility shift assays (EMSA).

Six psoralen-conjugated oligonucleotides were found to selectively and efficiently intercalate to their bovine dsDNA target sequences (38-49 bp) in vitro after irradiation with UV light (360 nm), forming covalent DNA photoadducts. Stable photoadducts were also detected when these oligonucleotides were targeted to larger dsDNA targets, ranging from 0.1 to 3 Kb in size. In general, a small UV dose (0.03 J/cm²) was sufficient to form monoadducts, with crosslinks forming after fifteen minutes of UV irradiation (1 J/cm²). The efficiency of bovine monoadduct formation was similar to that in the well-publicised supF DNA triplex system.

PCR and Southern blot analyses also showed that the repetitive Y-chromosomal DNA region (100-400 bp) was indeed male specific and present in multiple copies. However, when Southern blot analyses were carried out using oligonucleotide probes (16-49 nt) which corresponded to the triplex target sequence within this region, relatively weak hybridisation was observed, suggesting that these specific sequences were not present in such high copies. Although this data indicates that the male specific repeat regions must contain segments of consensual rather than exact sequences, high quality non-degenerate DNA sequencing data in the triplex targeting region was obtained from the male specific PCR products. Clearly, the exact nature of these male specific repeats requires further investigation to establish the effects of targeting psoralen-conjugated oligonucleotides to male genomic DNA as opposed to female genomic DNA.

year: 1999

title: The six days and the deluge: some ideas on earth history in the Royal Society of London 1660-1775

authors: Stokes, Evelyn

abstract: The influence of the biblical story of Creation and the Deluge on ideas of earth history during the period 1660-1775 is examined with particular reference to papers on the subject published in the Philosophical Transactions of the Royal Society of London. Topics examined in more detail include the controversies over the origin of marine fossils and bones of prehistoric animals, ideas on natural causes of the Deluge and its role in shaping landforms, and the age of the earth. Despite the inhibiting effect of the Genesis account, there was considerable flexibility in interpretation of both the Creation and Deluge stories in terms of current scientific knowledge. The later papers display a good deal of uniformitarian thinking within the framework of a catastrophic deluge hypothesis.

text: The six days and the deluge: some ideas on earth history in the Royal Society of London 1660-1775 The influence of the biblical story of Creation and the Deluge on ideas of earth history during the period 1660-1775 is examined with particular reference to papers on the subject published in the Philosophical Transactions of the Royal Society of London. Topics examined in more detail include the controversies over the origin of marine fossils and bones of prehistoric animals, ideas on natural causes of the Deluge and its role in shaping landforms, and the age of the earth. Despite the inhibiting effect of the Genesis account, there was considerable flexibility in interpretation of both the Creation and Deluge stories in terms of current scientific knowledge. The later papers display a good deal of uniformitarian thinking within the framework of a catastrophic deluge hypothesis.

year: 1969

title: The use of Māori words in National Science Challenge online discourse

authors: Calude, Andreea S.

subjects: Maori loanwords

abstract: New Zealand English is well-known for its heavy borrowing of words from Māori. This lexical component, unique to New Zealand English alone has been studied intensely over the last 50 years, particularly in newspaper media. Current research suggests the use is still increasing today, primarily in Māori-related contexts. Here, we analyse a surprising and unexpected use of Māori loanwords in science digital discourse (neither genre being previously investigated), where we discover a strong presence of Māori borrowings in National Science Challenge website and Twitter content. Using corpus linguistics methods, we argue that the use of Māori loanwords in this genre functions as a national identity building tool, used by various authors to signal that the ‘challenges’ the country faces are uniquely New Zealand’s ‘challenges’.

text: The use of Māori words in National Science Challenge online discourse New Zealand English is well-known for its heavy borrowing of words from Māori. This lexical component, unique to New Zealand English alone has been studied intensely over the last 50 years, particularly in newspaper media. Current research suggests the use is still increasing today, primarily in Māori-related contexts. Here, we analyse a surprising and unexpected use of Māori loanwords in science digital discourse (neither genre being previously investigated), where we discover a strong presence of Māori borrowings in National Science Challenge website and Twitter content. Using corpus linguistics methods, we argue that the use of Māori loanwords in this genre functions as a national identity building tool, used by various authors to signal that the ‘challenges’ the country faces are uniquely New Zealand’s ‘challenges’.

year: 2019