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Specificity and functionality of microRNA inhibitors.

Robertson B, Dalby AB, Karpilow J, Khvorova A, Leake D, Vermeulen A - Silence (2010)

Bottom Line: Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function.The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dharmacon Products, Thermo Fisher Scientific, 2650 Crescent Drive, Suite 100 Lafayette, CO 80026, USA. annaleen.vermeulen@thermofisher.com.

ABSTRACT

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.

No MeSH data available.


Crossreactivity is evident between let-7 microRNA family inhibitors. (a) Sequences from miRBase http://www.mirbase.org/ of the nine let-7 family members studied in this experiment. The 'seed' region (nucleotides 2 to 8), is indicated by shading. Nucleotides at which other family members differ from let-7a are underlined and in bold. (b) The let-7a dual-luciferase reporter was co-transfected with the negative control (NC, an equal concentration of non-functional nucleic acid molecule) or inhibitors targeting let-7a, let-7b or let-7c. (c) let-7b dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7 c. (d) A let-7c dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7c. HeLa cells were co-transfected with reporters and inhibitors 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Inhibitor concentrations ranged from 0.17 to 21 nM; plasmid concentrations were constant at 100 ng/well. Dual-luciferase ratios were measured 2 days post-transfection. Results shown are averages from triplicate wells, normalized to appropriate controls, then expressed as fold-inhibition relative to negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
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Figure 1: Crossreactivity is evident between let-7 microRNA family inhibitors. (a) Sequences from miRBase http://www.mirbase.org/ of the nine let-7 family members studied in this experiment. The 'seed' region (nucleotides 2 to 8), is indicated by shading. Nucleotides at which other family members differ from let-7a are underlined and in bold. (b) The let-7a dual-luciferase reporter was co-transfected with the negative control (NC, an equal concentration of non-functional nucleic acid molecule) or inhibitors targeting let-7a, let-7b or let-7c. (c) let-7b dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7 c. (d) A let-7c dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7c. HeLa cells were co-transfected with reporters and inhibitors 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Inhibitor concentrations ranged from 0.17 to 21 nM; plasmid concentrations were constant at 100 ng/well. Dual-luciferase ratios were measured 2 days post-transfection. Results shown are averages from triplicate wells, normalized to appropriate controls, then expressed as fold-inhibition relative to negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.

Mentions: To gain insight into the level of inhibitor crossreactivity to be expected between closely related family members, hairpin inhibitors (see Methods) designed against each of the nine human let-7 miRNAs (Figure 1a) were chosen for study. Some human let-7 miRNAs are expressed in many common immortal cell lines. The nine family members have sequences that differ from the canonical let-7a at either single or multiple nucleotide positions (Figure 1a). The assay system used was a set of dual-luciferase reporters for each of the let-7 miRNAs, as this type of reporter has demonstrated sufficient sensitivity to distinguish between inhibitors with only slight differences in functionality [20]. The target sites in these reporters are perfectly complementary to the mature miRNAs, because mismatched/attenuation type target sites were found to be much less sensitive [20]. All possible inhibitor/reporter pairs were tested by co-transfection into HeLa cells. The results clearly demonstrated that human let-7 miRNA inhibitors and reporter constructs, either alone or in combination, are non-specific (Figure 1b-d; also see Additional file 1, Figure S1). For each reporter, all inhibitors at 20 nM caused detectable fold changes in luciferase signal relative to the negative control. However, there was no consensus on crossreactivity ranking. For example, in both the let-7a and let-7c reporter assays, the let-7a and let-7c inhibitors caused a similar response in luciferase signal (approximately ninefold increase at 20 nM), whereas the let-7b inhibitor caused a lower response (approximately five-fold increase at 20 nM) (Figure 1b, Figure 1d). These data imply that the let-7a and let-7c inhibitors crossreact equally with each other, whereas the let-7b inhibitor has lower crossreactivity. However, in the let-7b reporter assay, the effects of the let-7a and let-7b inhibitors were similar (approximately sevenfold increase at 20 nM), whereas the effects of the let-7c inhibitor were much greater (approximately 13-fold increase at 20 nM) (Figure 1c). These latter data suggest that there is equivalent crossreactivity between the let-7a and the let-7b inhibitors, and leaves the let-7c crossreactivity open to interpretation. One plausible explanation is that multiple let-7 miRNAs are present in HeLa cells at varying concentrations and that both inhibitors and luciferase reporters crossreact with these miRNAs. The data also suggest that reporters (expressed mRNAs) and inhibitors (synthetic modified oligonucleotides) have different criteria for crossreactivity with endogenous miRNAs. Although it is possible to specifically detect expression of mature miRNAs [21-24], quantifying the level of functionally active mature miRNA (to measure inhibitor function) for closely related sequences is technically challenging. Owing to the difficulties inherent in working with multiple, related, endogenously expressed miRNAs, we decided to develop an assay in a less complex system.


Specificity and functionality of microRNA inhibitors.

Robertson B, Dalby AB, Karpilow J, Khvorova A, Leake D, Vermeulen A - Silence (2010)

Crossreactivity is evident between let-7 microRNA family inhibitors. (a) Sequences from miRBase http://www.mirbase.org/ of the nine let-7 family members studied in this experiment. The 'seed' region (nucleotides 2 to 8), is indicated by shading. Nucleotides at which other family members differ from let-7a are underlined and in bold. (b) The let-7a dual-luciferase reporter was co-transfected with the negative control (NC, an equal concentration of non-functional nucleic acid molecule) or inhibitors targeting let-7a, let-7b or let-7c. (c) let-7b dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7 c. (d) A let-7c dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7c. HeLa cells were co-transfected with reporters and inhibitors 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Inhibitor concentrations ranged from 0.17 to 21 nM; plasmid concentrations were constant at 100 ng/well. Dual-luciferase ratios were measured 2 days post-transfection. Results shown are averages from triplicate wells, normalized to appropriate controls, then expressed as fold-inhibition relative to negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
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Related In: Results  -  Collection

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Figure 1: Crossreactivity is evident between let-7 microRNA family inhibitors. (a) Sequences from miRBase http://www.mirbase.org/ of the nine let-7 family members studied in this experiment. The 'seed' region (nucleotides 2 to 8), is indicated by shading. Nucleotides at which other family members differ from let-7a are underlined and in bold. (b) The let-7a dual-luciferase reporter was co-transfected with the negative control (NC, an equal concentration of non-functional nucleic acid molecule) or inhibitors targeting let-7a, let-7b or let-7c. (c) let-7b dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7 c. (d) A let-7c dual-luciferase reporter was co-transfected with NC or inhibitors targeting let-7a, let-7b or let-7c. HeLa cells were co-transfected with reporters and inhibitors 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Inhibitor concentrations ranged from 0.17 to 21 nM; plasmid concentrations were constant at 100 ng/well. Dual-luciferase ratios were measured 2 days post-transfection. Results shown are averages from triplicate wells, normalized to appropriate controls, then expressed as fold-inhibition relative to negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
Mentions: To gain insight into the level of inhibitor crossreactivity to be expected between closely related family members, hairpin inhibitors (see Methods) designed against each of the nine human let-7 miRNAs (Figure 1a) were chosen for study. Some human let-7 miRNAs are expressed in many common immortal cell lines. The nine family members have sequences that differ from the canonical let-7a at either single or multiple nucleotide positions (Figure 1a). The assay system used was a set of dual-luciferase reporters for each of the let-7 miRNAs, as this type of reporter has demonstrated sufficient sensitivity to distinguish between inhibitors with only slight differences in functionality [20]. The target sites in these reporters are perfectly complementary to the mature miRNAs, because mismatched/attenuation type target sites were found to be much less sensitive [20]. All possible inhibitor/reporter pairs were tested by co-transfection into HeLa cells. The results clearly demonstrated that human let-7 miRNA inhibitors and reporter constructs, either alone or in combination, are non-specific (Figure 1b-d; also see Additional file 1, Figure S1). For each reporter, all inhibitors at 20 nM caused detectable fold changes in luciferase signal relative to the negative control. However, there was no consensus on crossreactivity ranking. For example, in both the let-7a and let-7c reporter assays, the let-7a and let-7c inhibitors caused a similar response in luciferase signal (approximately ninefold increase at 20 nM), whereas the let-7b inhibitor caused a lower response (approximately five-fold increase at 20 nM) (Figure 1b, Figure 1d). These data imply that the let-7a and let-7c inhibitors crossreact equally with each other, whereas the let-7b inhibitor has lower crossreactivity. However, in the let-7b reporter assay, the effects of the let-7a and let-7b inhibitors were similar (approximately sevenfold increase at 20 nM), whereas the effects of the let-7c inhibitor were much greater (approximately 13-fold increase at 20 nM) (Figure 1c). These latter data suggest that there is equivalent crossreactivity between the let-7a and the let-7b inhibitors, and leaves the let-7c crossreactivity open to interpretation. One plausible explanation is that multiple let-7 miRNAs are present in HeLa cells at varying concentrations and that both inhibitors and luciferase reporters crossreact with these miRNAs. The data also suggest that reporters (expressed mRNAs) and inhibitors (synthetic modified oligonucleotides) have different criteria for crossreactivity with endogenous miRNAs. Although it is possible to specifically detect expression of mature miRNAs [21-24], quantifying the level of functionally active mature miRNA (to measure inhibitor function) for closely related sequences is technically challenging. Owing to the difficulties inherent in working with multiple, related, endogenously expressed miRNAs, we decided to develop an assay in a less complex system.

Bottom Line: Synthetic miRNA target analogs, which are fully complementary, chemically modified oligonucleotides, have been used successfully to inhibit miRNA function.The results showed that the function of inhibitors vary as mismatch positions in the inhibitors change.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dharmacon Products, Thermo Fisher Scientific, 2650 Crescent Drive, Suite 100 Lafayette, CO 80026, USA. annaleen.vermeulen@thermofisher.com.

ABSTRACT

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.

No MeSH data available.