Specificity and functionality of microRNA inhibitors

Background: Micro(mi)RNAs regulate gene expression through translational attenuation and messenger (m)RNA degradation, and are associated with differentiation, homeostasis and disease. Natural miRNA target recognition is determined primarily by perfect complementarity in a seed region (nucleotide positions 2 to 7) with additional interactions contributing in a sequence- and target-specific manner. Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function. Results: In this paper, we present a first systematic study to evaluate the effect of mismatches in the target site on synthetic inhibitor activity. Panels of miRNA inhibitors containing two-nucleotide mismatches across the target site were tested against three miRNAs (miR-21, miR-22 and miR-122). The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change. Conclusions: The data indicate that features important for natural miRNA target recognition (such as seed region complementarity) are also important for inhibitor functionality. In addition, base pairing at a second, more 3 ' region appears to be equally important in determining the efficacy of synthetic inhibitors. Considering the importance of these inhibitor regions and the expression of closely related miRNA sequences will enable researchers to interpret results more accurately in future experiments

Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

BACKGROUND:Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells. CONCLUSIONS/SIGNIFICANCE:Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3\u27-CH2-CO-NH-5\u27 amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5\u27-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications

A complex containing CstF-64 and the SL2 snRNP connects mRNA 3′ end formation and trans-splicing in C. elegans operons

Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3′ end formation of the upstream mRNA and SL2-specific trans-splicing of the downstream mRNA. These processes usually occur within an ∼100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegans extracts containing the 3′ end formation protein CstF-64 and the SL2 snRNP. This complex, immunoprecipitated with αCstF-64 antibody, contains SL2 RNA, but not SL1 RNA or other U snRNAs. Using mutational analysis we have been able to uncouple SL2 snRNP function and identity. SL2 RNA with a mutation in stem/loop III is functional in vivo as a trans-splice donor, but fails to splice to SL2-accepting trans-splice sites, suggesting that it has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutations prevent association of SL2 RNA with CstF-64. In contrast, a mutation in stem II that inactivates the SL2 snRNP still permits complex formation with CstF-64. Therefore, SL2 RNA stem/loop III is required for both SL2 identity and formation of a complex containing CstF-64, but not for trans-splicing. These results provide a molecular framework for the coupling of 3′ end formation and trans-splicing in the processing of polycistronic pre-mRNAs from C. elegans operons

Percent of successful builds of DNA constructs with varying GC content.

<p>The bar graph is a visual representation of the data in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.t002" target="_blank">2</a>. Blue bars—represent the successful builds with the standard protocol. Red bars—represent the successful builds with the broad spectrum protocol.</p

PCR amplification of high and low GC oligonucleotides with and without subcycling and varying additives.

<p>A. 10% GC, no sub-cycling. B. 79% GC, no sub-cycling. C. 10% GC, sub-cycling. D. 79% GC, sub-cycling. First lane on the left of each gel: MW markers. Lanes numbered as follows: 1) no additive 2) 40:60 dGTP:deaza-dGTP 3) 50:50 dGTP:deaza-dGTP 4) 60:40 dGTP:deaza-dGTP 5) 2.5% DMSO 6) 10% DMSO 7) 0.1M betaine 8) 0.2M betaine 9) 0.4M betaine 10) 50:50 dGTP:deaza-dGTP with 5% DMSO 11) 60:40 dGTP:deaza-dGTP with 10% DMSO. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.</p

Schematics of the subcycling PCR protocol.

<p>A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.</p

PCR amplification of oligonucleotides with wide range GC contents with subcycling and different additives.

<p>A. No additives; B. 60% deaza-dGTP; C. 0.2M betaine. First lane on the left of each gel: MW markers. Lanes numbered indicates oligonucleotides of varying GC content as follows: 1) 10% GC; 2) 21%GC; 3) 33%GC; 4) 44%GC; 5) 56%GC; 6) 67%GC; 7) 79% GC; 8) 90%GC. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows expected PCR pattern where a strong 200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.</p

Percent of successful builds of DNA constructs with varying GC content using the standard protocol.

<p>The same data is visualized as a bar graph in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156478#pone.0156478.g005" target="_blank">Fig 5</a>.</p