Richard T. Pon

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin–proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

A long chain biotin phosphoramidite reagent for the automated synthesis of 5′-biotinylated oligonucleotides

Abstract An N-dimethoxytritylated biotin group coupled to a 6-aminohexanol linker was converted into a phosphoramidite to yield a biotinylating reagent which can be used in automated solid-phase synthesis to produce 5′-biotinylated oligonucleotides in high yields.

Hydroquinone-O,O′-diacetic acid as a more labile replacement for succinic acid linkers in solid-phase oligonucleotide synthesis

Abstract Hydroquinone-O,O′-diacetic acid (QDA) can be used to link nucleosides to CPG or polystyrene supports instead of succinic acid. Cleavage of oligodeoxy- or oligoribonucleotides, using ammonium hydroxide, requires only two to five minutes. The QDA linker is stable, easily prepared and does not require any other changes to the reagents, methods, or instrumentation used in automated solid-phase oligonucleotide synthesis.

Enhanced coupling efficiency using 4-dimethylaminopyridine (DMAP) and either tetrazole, 5-(o-nitrophenyl) tetrazole, or 5-(p-nitrophenyl) tetrazole in the solid phase synthesis of oligoribonucleotides by the phosphoramidite procedure.

Abstract 5-( p -nitrophenyl) tetrazole/DMAP mixtures are more effective phosphoramidite activators than either tetrazole, 5-( o -nitrophenyl) tetrazole or 5-( p -nitrophenyl) tetrazole for solid phase oligonucleotide synthesis.

N-phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase synthesis of oligoribonucleotides: synthesis of a ribozyme sequence

Abstract Phosphoramidites for guanosine and adenosine nucleosides with phenoxyacetyl as N-acyl protecting group were prepared. These nucleoside phosphoramidites, together with previously reported uridine and N-benzoyl cytidine phosphoramidites have been applied to the efficient solid phase synthesis of a trideca and a nonadecaribonucleotide. The later molecule has the sequence corresponding to a ribozyme.

Rapid automated derivatization of solid-phase supports for oligonucleotide synthesis using uronium or phosphonium coupling reagents

Abstract Very fast (4 sec) attachment of nucleosides to CPG supports, via succinyl or QDA linkers, is possible with either uronium (HBTU, HATU, HBPyU, HBPipU) or phosphonium (BOP, PyBOP ® , PyBroP, BroP) coupling reagents and automated derivatization of CPG and polystyrene supports just prior to oligonucleotide synthesis has been demonstrated.

PROPERTIES OF ARABINONUCLEIC ACIDS (ANA & 20′F-ANA): IMPLICATIONS FOR THE DESIGN OF ANTISENSE THERAPEUTICS THAT INVOKE RNASE H CLEAVAGE OF RNA

Inversion of configuration of the C2′ position of RNA leads to a very unique nucleic acid structure: arabinonucleic acid (ANA). ANA, and its 2′-fluoro derivative (2′ F-ANA) form hybrids with RNA that are capable of activating RNase H, resulting in cleavage of the RNA strand. In this paper, we review the properties of duplexes formed between ANA (or 2′F-ANA) and its RNA complement. These studies support the notion that RNase H is sensitive to the minor groove dimensions of the hybrid substrate.

Inhibition of cell adhesion to plastic substratum by phosphorothioate oligonucleotide.

Antisense oligonucleotides have been widely used to achieve specific inhibition of targeted gene expression. However, the mechanism of action is not well understood and in many systems sequence-independent effects occur. We have recently shown that chronic administration of an antisense c-myc phosphorothioate oligonucleotide can specifically inhibit expression of the c-myc protein and growth in human breast cancer cells. We now identify an additional effect of the same oligonucleotide on cell adhesion. Transient delivery through electroporation of 2.5 microM antisense-myc oligonucleotide to MCF-7 cells results in 85% inhibition of adhesion to plastic substratum within 24 h. Both the onset of this effect and the subsequent recovery occur without a change in cell viability, growth, or alteration of adhesion to Matrigel, collagen IV, laminin, or fibronectin. However, no parallel changes in c-myc mRNA or protein expression are detectable, suggesting that in this instance inhibition of adhesion caused by antisense-myc oligonucleotide may involve a mechanism independent of the target sequence.