A digitally driven manufacturing process for high resolution patterning of cell formations.

This paper presents the engineering and validation of an enabling technology that facilitates new capabilities in in vitro cell models for high-throughput screening and tissue engineering applications. This is conducted through a computerized system that allows the design and deposition of high-fidelity microscale patterned coatings that selectively alter the chemical and topographical properties of cell culturing surfaces. Significantly, compared to alternative methods for microscale surface patterning, this is a digitally controlled and automated process thereby allowing scientists to rapidly create and explore an almost infinite range of cell culture patterns. This new capability is experimentally validated across six different cell lines demonstrating how the precise microscale deposition of these patterned coatings can influence spatiotemporal growth and movement of endothelial, fibroblast, neuronal and macrophage cells. To further demonstrate this platform, more complex patterns are then created and shown to guide the behavioral response of colorectal carcinoma cells.

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site.

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.

Inhibition of the osteoclast V-ATPase by small interfering RNAs.

The multisubunit enzyme V-ATPase harbours isoforms of individual subunits. a3 is one of four 116 kDa subunit a isoforms, and it is crucial for bone resorption. We used small interfering RNA (siRNA) molecules to knock down a3 in rat osteoclast cultures. Labeled siRNA-molecules entered osteoclasts via endocytosis and knocked down the a3 mRNA. Bone resorption was decreased in siRNA-treated samples due to decreased acidification and osteoclast inactivation. Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 in osteoclast cultures. Subunit a3 is thus an interesting target for novel nucleic acid therapy.

Bioinformatic approaches to siRNA selection and optimization.

RNA interference mediated by short interfering RNA (siRNA) molecules represents a powerful genetic tool with an increasing interest as potential therapeutics. Current bioinformatic approaches to design functional siRNA molecules take into account both empirical and rational approaches to identify selectable characteristics of active and specific siRNA molecules and focusing the downstream events in the RNAi pathway, such as target messenger RNA accessibility. The design of effective siRNA molecules is the key to successful experimentation with RNAi. Here, we show advanced siRNA design parameters and options for highly efficient siRNA candidate search.

Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules.

OBJECTIVE: Here we report the improved results of a new siRNA design program and analysis tool called siRNA_profile that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences. METHODS: We retrospectively analysed over 2400 siRNA sequences from 34 genes and with known efficacies to categorize factors that differentiate highly, moderately and non-functional siRNA sequences in more detail. We tested the biological relevance of siRNA_profile in CHO cells stably expressing human TRACP. RESULTS: The highly functional siRNA molecules exhibited lower overall stabilities than non-functional siRNAs after taking into consideration all the nucleotides from 5'-terminus to the 3'-terminus along the siRNA molecule, in addition to the 5'-section of the antisense strand and the region between 9-14 nucleotides as previously has been acknowledged. Comparison of the siRNA_profile program to five other programs resulted in a wide range of selected siRNA sequences with diverse gene silencing capacities, even when the target was only 197 nucleotides long. Six siRNA design programs selected 24 different siRNA sequences, and only 6 of them were selected by two or more programs. The other 18 sequences were individually selected by these six programs. CONCLUSION: Low general stability of dsRNA plays a significant role in the RNAi pathway and is a recommended criterion to consider, in addition to 5'-instability, internal instability, nucleotide preferences and target mRNA position, when designing highly efficient siRNAs.

Sequence and TLR9 independent increase of TRACP expression by antisense DNA and siRNA molecules.

UNLABELLED: Reactive oxygen species generating activity of tartrate-resistant acid phosphatase (TRACP) has been suggested to have several functions in TRACP expressing bone resorbing osteoclasts, macrophages, and dendritic cells. This work aimed to study the TRACP knock down phenotype in osteoclasts by using antisense DNA and RNA interference methods. Unexpectedly, both TRACP specific DNA oligonucleotides and siRNA molecules extensively increased the TRACP expression in human osteoclasts and monocytes. Toll-like receptor 9 (TLR9) is an immunity sensor for CpG motifs in DNA. We cultured bone marrow-derived osteoclast precursor cells from wild-type and TLR9-/- mice with CpG and non-CpG DNA oligonucleotides, and observed that the increased TRACP expression was sequence and TLR9 independent. In contrast, cells with increased TRACP activity showed decreased activity of tartrate-sensitive acid phosphatases. CONCLUSION: DNA oligonucleotides and RNA molecules extensively increase TRACP expression in monocyte-macrophage lineage. These results suggest a potential role of TRACP in pathogen recognition and in innate immunity.