Advancing peptide siRNA-carrier designs through L/D-amino acid stereochemical modifications to enhance gene silencing.

The 599 peptide has been previously shown to effectively deliver small interfering RNAs (siRNAs) to cancer cells, inducing targeted-oncogene silencing, with a consequent inhibition of tumor growth. Although effective, this study was undertaken to advance the 599 peptide siRNA-carrier design through L/D-amino acid stereochemical modifications. Consequently, 599 was modified to generate eight different peptide variants, incorporating either different stereochemical patterns of L/D-amino acids or a specific D-amino acid substitution. Upon analysis of the variants, it was observed that these modifications could, in some instances, increase/decrease the binding, nuclease/serum stability, and complex release of siRNAs, as well as influence the gene-silencing efficiencies of the complex. These modifications were also found to affect cellular uptake and intracellular localization patterns of siRNA cargo, with one particular variant capable of mediating binding of siRNAs to specific cellular projections, identified as filopodia. Interestingly, this variant also exhibited the most enhanced gene silencing in comparison to the parent 599 peptide, thus suggesting a possible connection between filopodia binding and enhanced gene silencing. Together, these data demonstrate the utility of peptide stereochemistry, as well as the importance of a key D-amino acid modification, in advancing the 599 carrier design for the enhancement of gene silencing in cancer cells.

Peptide carriers to the rescue: overcoming the barriers to siRNA delivery for cancer treatment.

Cancer is a significant health concern worldwide and its clinical treatment presents many challenges. Consequently, much research effort has focused on the development of new anticancer drugs to combat this disease. One area of exploration, in particular, has been in the therapeutic application of RNA interference (RNAi). Although RNAi appears to be an attractive therapeutic tool for the treatment of cancer, one of the primary obstacles towards its pervasive use in the clinic has been cell/tissue type-specific cytosolic delivery of therapeutic small interfering RNA (siRNA) molecules. Consequently, varied drug delivery platforms have been developed and widely explored for siRNA delivery. Among these candidate drug delivery systems, peptides have shown great promise as siRNA carriers due to their varied physiochemical properties and functions, simple formulations, and flexibility in design. In this review, we will focus on distinguishing between the different classes of peptide carriers based on their functions, as well as summarize and discuss the various design strategies and advancements that have been made in circumventing the barriers to siRNA delivery for cancer treatment. Resolution of these challenges by peptide carriers will accelerate the translation of RNAi-based therapies to the clinic.

Long noncoding RNA complementarity and target transcripts abundance.

Eukaryotic mRNA metabolism regulates its stability, localization, and translation using complementarity with counter-part RNAs. To modulate their stability, small and long noncoding RNAs can establish complementarity with their target mRNAs. Although complementarity of small interfering RNAs and microRNAs with target mRNAs has been studied thoroughly, partial complementarity of long noncoding RNAs (lncRNAs) with their target mRNAs has not been investigated clearly. To address that research gap, our lab investigated whether the sequence complementarity of two lncRNAs, lincRNA-p21 and OIP5-AS1, influenced the quantity of target RNA expression. We predicted a positive correlation between lncRNA complementarity and target mRNA quantity. We confirmed this prediction using RNA affinity pull down, microarray, and RNA-sequencing analysis. In addition, we utilized the information from this analysis to compare the quantity of target mRNAs when two lncRNAs, lincRNA-p21 and OIP5-AS1, are depleted by siRNAs. We observed that human and mouse lincRNA-p21 regulated target mRNA abundance in complementarity-dependent and independent manners. In contrast, affinity pull down of OIP5-AS1 revealed that changes in OIP5-AS1 expression influenced the amount of some OIP5-AS1 target mRNAs and miRNAs, as we predicted from our sequence complementarity assay. Altogether, the current study demonstrates that partial complementarity of lncRNAs and mRNAs (even miRNAs) assist in determining target RNA expression and quantity.

Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability.

Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post-transcription, and post-translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6-methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A-modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady-state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady-state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2-depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging.