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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.


Mismatched inhibitors show similar positional effects on the expression of an endogenous target. The effect of mismatches on miR-122 inhibitor function was determined by measuring steady-state levels of endogenous ALDOA mRNA. The set of miR-122 inhibitors [11 mismatched and one fully matched, and a negative control (NC) inhibitor] were transfected into Huh-7 cells 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Media was changed to new, antibiotic-free media approximately every 3 days. Response of ALDOA mRNA levels relative to a housekeeping gene was measured on day 6 (white bars) and day 9 (grey bars) post-transfection by branched-DNA assay. Results shown are averages from triplicate wells transfected with 4 nM inhibitor, normalized to appropriate controls, then expressed as fold-inhibition relative to transfection with the negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
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Figure 5: Mismatched inhibitors show similar positional effects on the expression of an endogenous target. The effect of mismatches on miR-122 inhibitor function was determined by measuring steady-state levels of endogenous ALDOA mRNA. The set of miR-122 inhibitors [11 mismatched and one fully matched, and a negative control (NC) inhibitor] were transfected into Huh-7 cells 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Media was changed to new, antibiotic-free media approximately every 3 days. Response of ALDOA mRNA levels relative to a housekeeping gene was measured on day 6 (white bars) and day 9 (grey bars) post-transfection by branched-DNA assay. Results shown are averages from triplicate wells transfected with 4 nM inhibitor, normalized to appropriate controls, then expressed as fold-inhibition relative to transfection with the negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.

Mentions: To determine whether the data observed in luciferase reporter assays could be extended to endogenous gene targets, we tested the effects of miR-122 inhibitors containing mismatches on the endogenous miR-122 target ALDOA [16-19,25]. The miR-122 mismatched inhibitors used in the reporter assays above were introduced into Huh-7 cells (doses ranging from 0.8 to 100 nM, data not shown) and mRNA levels of ALDOA were monitored. Differential responses between mismatched inhibitors were most evident for the measurements at 4 nM on day 6 (Figure 5). The mismatches producing the greatest decreases in inhibitor activity on endogenous mRNA levels (Figure 5, day 6) were those at positions 3 to 8 and 15 to 16, whereas mismatches at positions 1 to 2, 9 to 10 and 19 to 22 had the least effect on inhibitor activity compared with the fully matched inhibitor. A similar pattern was observed with the miR-122 luciferase assay (Figure 4b, 2 nM dose) suggesting that conclusions about inhibitor specificity based on reporter assays can be applied to inhibitor effects on endogenous targets.


Specificity and functionality of microRNA inhibitors.

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

Mismatched inhibitors show similar positional effects on the expression of an endogenous target. The effect of mismatches on miR-122 inhibitor function was determined by measuring steady-state levels of endogenous ALDOA mRNA. The set of miR-122 inhibitors [11 mismatched and one fully matched, and a negative control (NC) inhibitor] were transfected into Huh-7 cells 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Media was changed to new, antibiotic-free media approximately every 3 days. Response of ALDOA mRNA levels relative to a housekeeping gene was measured on day 6 (white bars) and day 9 (grey bars) post-transfection by branched-DNA assay. Results shown are averages from triplicate wells transfected with 4 nM inhibitor, normalized to appropriate controls, then expressed as fold-inhibition relative to transfection with the negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864222&req=5

Figure 5: Mismatched inhibitors show similar positional effects on the expression of an endogenous target. The effect of mismatches on miR-122 inhibitor function was determined by measuring steady-state levels of endogenous ALDOA mRNA. The set of miR-122 inhibitors [11 mismatched and one fully matched, and a negative control (NC) inhibitor] were transfected into Huh-7 cells 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. Media was changed to new, antibiotic-free media approximately every 3 days. Response of ALDOA mRNA levels relative to a housekeeping gene was measured on day 6 (white bars) and day 9 (grey bars) post-transfection by branched-DNA assay. Results shown are averages from triplicate wells transfected with 4 nM inhibitor, normalized to appropriate controls, then expressed as fold-inhibition relative to transfection with the negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
Mentions: To determine whether the data observed in luciferase reporter assays could be extended to endogenous gene targets, we tested the effects of miR-122 inhibitors containing mismatches on the endogenous miR-122 target ALDOA [16-19,25]. The miR-122 mismatched inhibitors used in the reporter assays above were introduced into Huh-7 cells (doses ranging from 0.8 to 100 nM, data not shown) and mRNA levels of ALDOA were monitored. Differential responses between mismatched inhibitors were most evident for the measurements at 4 nM on day 6 (Figure 5). The mismatches producing the greatest decreases in inhibitor activity on endogenous mRNA levels (Figure 5, day 6) were those at positions 3 to 8 and 15 to 16, whereas mismatches at positions 1 to 2, 9 to 10 and 19 to 22 had the least effect on inhibitor activity compared with the fully matched inhibitor. A similar pattern was observed with the miR-122 luciferase assay (Figure 4b, 2 nM dose) suggesting that conclusions about inhibitor specificity based on reporter assays can be applied to inhibitor effects on endogenous targets.

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.