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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.


Position of mismatches in the inhibitor target site affects inhibitor functionality. The effect of mismatches on inhibitor function was determined for (a) miR-21 in HeLa cells, (b) for miR-122 in Huh-7 cells and (c) for miR-22 in HeLa cells; cell lines were chosen for high expression of the respective miRNAs. Transfections were performed 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. For each microRNA, the appropriate dual-luciferase reporter, at 100 ng/well, was co-transfected with either a fully matched inhibitor or one of a set of 11 mismatched inhibitors at concentrations from 0.03 to 20 nM. 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 transfection with a negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
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Figure 4: Position of mismatches in the inhibitor target site affects inhibitor functionality. The effect of mismatches on inhibitor function was determined for (a) miR-21 in HeLa cells, (b) for miR-122 in Huh-7 cells and (c) for miR-22 in HeLa cells; cell lines were chosen for high expression of the respective miRNAs. Transfections were performed 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. For each microRNA, the appropriate dual-luciferase reporter, at 100 ng/well, was co-transfected with either a fully matched inhibitor or one of a set of 11 mismatched inhibitors at concentrations from 0.03 to 20 nM. 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 transfection with a negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.

Mentions: For all three miRNAs tested, mismatches causing greatest interference with inhibitor activity are located within positions corresponding to the seed region of the mature miRNAs (positions 3 to 8) and a second region closer to the 3' end (positions 13 to 18) (Figure 4). The purine and pyrmidine composition for the mismatches were examined to determine if mismatch identity affects the level of inhibition; no trend was observed in this set of data. However, for each of the three miRNAs, the exact positions within the two identified regions that were most deleterious for inhibitor function varied. The most detrimental mismatches within each miRNA were at positions 3 to 4 and 13 to 18 in miR-21 (Figure 4a; 20 nM), positions 3 to 8 and 15 to 16 in miR-122 (Figure 4b; 2 nM) and positions 5 to 8 and 13 to 16 in mIR-22 (Figure 4c; 0.3 nM). For all three miRNAs tested, mismatches located at the beginning (positions 1 and 2), end (positions 19 to 22) and middle (positions 9 and 10) of the inhibitor target site had the least effect on inhibitor function (Figure 4).


Specificity and functionality of microRNA inhibitors.

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

Position of mismatches in the inhibitor target site affects inhibitor functionality. The effect of mismatches on inhibitor function was determined for (a) miR-21 in HeLa cells, (b) for miR-122 in Huh-7 cells and (c) for miR-22 in HeLa cells; cell lines were chosen for high expression of the respective miRNAs. Transfections were performed 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. For each microRNA, the appropriate dual-luciferase reporter, at 100 ng/well, was co-transfected with either a fully matched inhibitor or one of a set of 11 mismatched inhibitors at concentrations from 0.03 to 20 nM. 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 transfection with a 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 4: Position of mismatches in the inhibitor target site affects inhibitor functionality. The effect of mismatches on inhibitor function was determined for (a) miR-21 in HeLa cells, (b) for miR-122 in Huh-7 cells and (c) for miR-22 in HeLa cells; cell lines were chosen for high expression of the respective miRNAs. Transfections were performed 1 day after plating into 96-well plates, 10,000 cells/well, in antibiotic-free media. For each microRNA, the appropriate dual-luciferase reporter, at 100 ng/well, was co-transfected with either a fully matched inhibitor or one of a set of 11 mismatched inhibitors at concentrations from 0.03 to 20 nM. 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 transfection with a negative control. Error bars are ± 1SD (sample) of the original triplicate data, scaled for all subsequent calculations.
Mentions: For all three miRNAs tested, mismatches causing greatest interference with inhibitor activity are located within positions corresponding to the seed region of the mature miRNAs (positions 3 to 8) and a second region closer to the 3' end (positions 13 to 18) (Figure 4). The purine and pyrmidine composition for the mismatches were examined to determine if mismatch identity affects the level of inhibition; no trend was observed in this set of data. However, for each of the three miRNAs, the exact positions within the two identified regions that were most deleterious for inhibitor function varied. The most detrimental mismatches within each miRNA were at positions 3 to 4 and 13 to 18 in miR-21 (Figure 4a; 20 nM), positions 3 to 8 and 15 to 16 in miR-122 (Figure 4b; 2 nM) and positions 5 to 8 and 13 to 16 in mIR-22 (Figure 4c; 0.3 nM). For all three miRNAs tested, mismatches located at the beginning (positions 1 and 2), end (positions 19 to 22) and middle (positions 9 and 10) of the inhibitor target site had the least effect on inhibitor function (Figure 4).

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.