LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem-loop hairpin oligonucleotide-a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.     

Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.     

Biochemical responses associated with resistance to bacterial stalk rot caused by Dickeya zeae in maize

Bacterial stalk rot (BSR) of maize caused by Dickeya zeae is an important disease in northwest region of India. In the current study, eighty maize lines were evaluated for resistance against this disease. Of these, 20 were moderately resistant, 25 were moderately susceptible and the rest were highly susceptible to BSR. Six lines from each set were randomly selected. Activities of three antioxidant enzymes, viz. phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO) were analysed from these three sets of maize lines representing different levels of resistance. A trend of elevated activity of PAL, POX and PPO was observed in all the three sets of maize lines. The results showed significantly more activity of these three enzymes in moderately resistant than highly susceptible maize lines. The activity of PAL and PPO peaked after 48 hr and of POX after 72 hr of challenge inoculation by D. zeae in all the maize lines. The activity of these enzymes further correlated negatively with disease development. Our results show that PAL, POX and PPO play an important role in contributing towards resistance in maize against BSR.     

Association of cyclin-dependent kinase 5 and its activator p35 with apoptotic cell death

We have investigated the role of cyclin-dependent kinases in cell death and found that the expression of cyclin-dependent kinase 5 (Cdk5) is associated with apoptotic cell death in both adult and embryonic tissues. By double labeling immunohistochemistry and confocal microscopy, we specifically associated the expression of Cdk5 to dying cells. The association of Cdks with cell death is unique to Cdk5 as this association is not found with the other Cdks (Cdk 1–8) and cell death. The differential increase in Cdk5 expression is at the level of protein only, and no differences can be detected at the level of mRNA. Using both limbs of mutant mice defective in the pattern of interdigital cell death and limbs with increased interdigital cell death by retinoic acid treatment, we confirmed the specificity of Cdk5 protein expression in dying cells. To investigate the regulation of Cdk5 during cell death, we examined the expression of a regulatory protein of Cdk5, p35, and found p35 to be expressed in the dying cells as well. Similar to Cdk5, there is also no specific differential expression of the p35 mRNA in dying cells. Our results suggest a role for Cdk5 and p35 proteins in cell death. This protein complex may function in the rearrangement of the cytoskeleton during apoptosis. Dev. Genet. 21:258–267, 1997. © 1997 Wiley-Liss, Inc.