Reading DNA differently

Product inhibition has provided the limiting barrier to efficient template-directed ligation and polymerization reactions. Here we review the attempts to circumvent this limitation and outline a traslation strategy that does overcome the barrier and allows the information encoded in DNA to be read and amplified into backbone-modified oligonucleotides. © 1998 John Wiley & Sons, Inc. Biopoly 48: 19–28, 1998     

Stabilization of G•C-Containing DNA Duplexes by Polyamides with Parallel Orientation in the Minor Groove

The polyamides based on 4-amino-1-methylpyrrol-2-carboxylic acid, 4-amino-1-methylimidazole-2-carboxylic acid, and -alanine that stabilize oligonucleotide duplexes consisting of GC pairs through parallel packing in the minor groove were studied. The initial duplex TTGCGCpGCGCAA melts at 28°C; the TTGCGCp[NH(CH₂)₃COPyImImNH(CH₂)₃NH(CH₃)₂][NH(CH₂)₃COImImPyNH(CH₂)₃N(CH₃)₂]GCGCAA duplex (bisphosphoramidate with parallel orientation of ligands, where Py, Im, and are the residues of 1-methyl-4-aminopyrrol-2-carboxylic and 1-methyl-4-aminoimidazole-2-carboxylic acids and -alanine, respectively), at 48°C; and the TTGCGCp[NH(CH₂)₃COImImPyNH(CH₂)₃COImImPyNH(CH₂)₃N(CH₃)₂]GCGCAA duplex (a hairpin structure with antiparallel orientation), at 56°C.     

AAV mediated expression of anti-sense neuropeptide Y cRNA in the arcuate nucleus of rats results in decreased weight gain and food intake

Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3 +/- 2.0 fmol/L [AS-NPY] vs 37.3 +/- 7.7 fmol/L [control], mean +/- SEM p < 0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42% +/- 6.5% [AS-NPY] vs 100% +/- 36% [control], 20 days 41% +/- 6% [AS-NPY] vs 100% +/- 27% [control] mean+/-SEM, p < 0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25 +/- 1.10 g [AS-NPY] vs 9.42 +/- 0.65 g [control] mean +/- SEM, p < 0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0 +/- 9.6 g [AS-NPY] vs 82.0 +/- 6.3 g [control] mean +/- SEM, p < 0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8 +/- 1.9 g [AS-NPY] vs 250.6 +/- 8.7 g [control], mean +/- SEM, p < 0.05) and remained so until the end of the study (day 50: 834.5 +/- 14.8 g [AS-NPY] vs 926.0 +/- 31.7 g [control], mean +/- SEM, p < 0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7-14: 24.9 +/- 0.4 g/day [AS-NPY] vs 27.2 +/- 0.4 g/day [control], mean +/- SEM, p < 0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.     

反义核酸药物的研究现状

反义药物以其合理设计药物的可能性和精确的特异性广泛吸引了人们的注意,但反义药物的研究并非如人们最初预想的那样简单。本文从其特异性,稳定性,透过靶细胞的能力,作用强度,活性判定,给药途径,安全性和毒性,生产成本等诸方面对反义药物的研究现状,现存问题进行了综述。相信伴随这些问题的解决,反义药物很可能成为药典的一部分,给疾病的治疗带来益处。     

Reaction parameters of targeted gene repair in mammalian cells

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.     

Sensitized site-specific photomodification of ssDNA by binary systems of oligonucleotide conjugates: VI. Effect of substituents in the sensitizer anthracene residue

Photomodification of ssDNA by binary systems of oligonucleotide conjugates complementary to the adjacent sequences of the target DNA was studied. One of the conjugates comprised a substituted anthracene as a sensitizer; the other,p-azidotetrafluorobenzaldehyde 3-aminopropionylhydrazone as a photoreagent. The sensitized photomodification is initiated by the 365–580-nm light through an efficient energy transfer from the photoexcitated sensitizer onto the photoreagent in a complementary complex of the binary system with the DNA target where the sensitizer and the photoreagent are sterically converged. Influence of substituents in the anthracene residue on the efficiency of the DNA sensitized photomodification was considered. The oligonucleotide conjugate of anthracene-9-al 3-aminopropionylhydrazone allows highly specific initiation of the sensitized photomodification upon irradiation with visible light at >460 nm in conditions generating no photoreaction in the sensitizer’s absence. For Part V, see [1]; prefix “d” in designations of oligonucleotides is omitted.     

Analogues of Uridinediphosphatehexoses. A New Type of Protein Glycosylation Inhibitors That Show Antiviral Activity

Abstract

A series of analogues of UDP-Glc and UDP-GlcNAc prepared by reaction of protected hexoses with ClSO₂NCO and 2′3′-O-isopropylideneuridine, inhibited glycosylation of proteins in HSV-1 infected HeLa cells and were active against several enveloped viruses.     

α-Hydroxyphosphonate Oligonucleotides: A Promising DNA Type?

Abstract

The synthesis of monomers ( S )-1, ( R )-1 and 2 derived from (5′ S )-, (5′ R )-2′-deoxythymidine-5′-C-phosphonic acids and 2′,5′-dideoxythymidine-5′-C-phosphonic acids was elaborated. The protection of the 5′-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers ( S )-3, ( S )-4, ( S )-5, ( R )-4 and ( R )-5 containing the chiral 3′-O-P-CH(OH)-5″ internucleotide linkage. Their hybridization properties with dA₁₅ and rA₁₅ were studied as well as their resistance against nuclease cleavage.     

Development Of The Novel Drug Releasing System Triggered By Hybridization With Target Sequence

We have already established the strategy of synchronous activation by hybridization, in which the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, can be generated from its stable precursors, the phenylsulfide derivatives, by a hybridization-promoted activation process with selectivity to cytosine. In this study, this in situ activation system was applied to the method for the drug releasing system triggered by hybridization with the target sequence.