Background

Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection.

Methodology

In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition.

Conclusions

We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers

We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°Nm, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs involved in the post-transcriptional gene regulation and play a critical role in plant growth, development and stresses response. However less is known about miRNAs involvement in grafting behaviors, especially with the watermelon (Citrullus lanatus L.) crop, which is one of the most important agricultural crops worldwide. Grafting method is commonly used in watermelon production in attempts to improve its adaptation to abiotic and biotic stresses, in particular to the soil-borne fusarium wilt disease. In this study, Solexa sequencing has been used to discover small RNA populations and compare miRNAs on genome-wide scale in watermelon grafting system. A total of 11,458,476, 11,614,094 and 9,339,089 raw reads representing 2,957,751, 2,880,328 and 2,964,990 unique sequences were obtained from the scions of self-grafted watermelon and watermelon grafted on-to bottle gourd and squash at two true-leaf stage, respectively. 39 known miRNAs belonging to 30 miRNA families and 80 novel miRNAs were identified in our small RNA dataset. Compared with self-grafted watermelon, 20 (5 known miRNA families and 15 novel miRNAs) and 47 (17 known miRNA families and 30 novel miRNAs) miRNAs were expressed significantly different in watermelon grafted on to bottle gourd and squash, respectively. MiRNAs expressed differentially when watermelon was grafted onto different rootstocks, suggesting that miRNAs might play an important role in diverse biological and metabolic processes in watermelon and grafting may possibly by changing miRNAs expressions to regulate plant growth and development as well as adaptation to stresses. The small RNA transcriptomes obtained in this study provided insights into molecular aspects of miRNA-mediated regulation in grafted watermelon. Obviously, this result would provide a basis for further unravelling the mechanism on how miRNAs information is exchanged between scion and rootstock in grafted watermelon, and its relevance to diverse biological processes and environmental adaptation.

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.