“Locked onto the target”: increasing the efficiency of antagomirzymes using locked nucleic acid modifications

This study highlights the effect of incorporation of locked nucleic acid (LNA) on improving the functional efficacy of DNAzymes against microRNAs (antagomirzymes). DNAzymes were designed against two different sites of miR-27a, which were encompassed both within the precursor and mature form of miRNA. The cleavage and functional activities of these DNAzymes have been compared to those of LNA-modified counterparts, containing LNA modification in each of the substrate binding arms. Preliminary examination based on in vitro cleavage demonstrated LNAzyme to be much more effective in the ensuing cleavage of target miRNA under both single- and multiple-turnover conditions. Evaluation of kinetic parameters indicated almost 5-fold higher cleavage efficiency, kobs, for LNAzymes than for DNAzymes, leading to more efficient cleavage of the substrate. We attribute this enhancement in cleavage efficiency to the LNA-mediated improvement in the hybridization of the antagomirzyme·target complex. Functional validation of the relative activities was accomplished through the luciferase reporter assay and quantitative real-time polymerase chain reaction (qRT-PCR). Both the unmodified and LNA-modified antagomirzymes were very active in ensuing efficient miRNA knockdown; however, compared to the DNAzymes, the LNAzymes were almost 25% more active. A direct quantitative estimate of miRNA cleavage, conducted using qRT-PCR, further substantiated the data by indicating that LNAzyme effectively downregulated the levels of mature miRNA (up to 50%) versus the corresponding DNAzymes. Our data thus provide formative evidence of the successful employment of LNA-based antagomirzymes against miRNA.     

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.