LidNA, a novel miRNA inhibitor constructed with unmodified DNA

FEBS Letters 586 (2012) 1529–1532

journal homepage: www.FEBSLetters.org

LidNA, a novel miRNA inhibitor constructed with unmodified DNA Akira Tachibana ⇑, Yui Yamada, Hiroyuki Ida, Satoshi Saito, Toshizumi Tanabe Department of Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan

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Article history: Received 2 February 2012 Revised 21 March 2012 Accepted 11 April 2012 Available online 20 April 2012 Edited by Tamas Dalmay Keywords: miRNA inhibitor miRNA RNAi SPR Unmodified DNA RISC

a b s t r a c t Many miRNA inhibitors have been developed and they are chemically modified oligonucleotides such as 20 -O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA was not yet reported as a miRNA inhibitor because of the low affinity of DNA/miRNA compared to mRNA/miRNA. We designed a structured unmodified DNA that significantly inhibits miRNA function. The clue structure for activity is the miRNA binding site between double stranded regions which is responsible for the miRNA inhibitory activity and tight binding to miRNA. We developed the miRNA inhibitor constructed with unmodified DNA, and named it LidNA, DNA that puts a lid on miRNA function. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction miRNAs are a class of endogenously expressed small regulatory non-coding RNAs thought to negatively regulate target mRNAs by binding with imperfect complementarity in their 30 -untranslated regions (30 -UTR) [1,2]. The diverse nature of miRNA target sites is reflected in the results of both experimentally validated and computationally based mirna target site predictions, which suggest that, on average, each miRNA targets hundreds of genes [3,4]. miRNAs have been demonstrated to play roles in cell differentiation and proliferation. The transfection of miR-302s expression vector to cells, without the other protein expression vector, led to the reprograming of human hair follicle cells into a human embryonic stem celllike pluripotent state [5,6]. Some miRNAs show differential expression levels in cancer and are able to affect cellular transformation, carcinogenesis and metastasis, acting either as oncogenes or tumor suppressors [7]. MiR-21, for example, is an unique miRNA in that it is overexpressed in most tumor types analysed so far. Moreover, the overexpression of miR-21 was reported that leads to a pre-B malignant lympolid-like phenotype, demonstrating that mir-21 is a genuine oncogene [8]. Current biochemically based tools for the sequence-specific inhibition of miRNA function are limited to single-stranded oligonucleotide-based miRNA inhibitors. These molecules are fully Abbreviations: miRNA, microRNA; 30 -UTR, 30 -untranslated region; LNA, locked nucleic acid; RISC, RNA-induced silencing complex ⇑ Corresponding author. E-mail address: [email protected] (A. Tachibana).

20 -O-methylated reverse complements of the mature miRNA sequences [9–12] and are believed to act as non-cleavable substrates (antisense) of the RNA-induced silencing complex (RISC). The miRNA inhibitory effects of alternative chemical modification patterns including locked nucleic acids (LNA), phosphorothioate and 20 -methoxyethyl modifications, have also been investigated [13–15]. DNA vectors that express ‘microRNA sponge’ and ‘tough decoy RNA’ as the competitive inhibitor of miRNA have been described [16,17]. These expressed RNAs have mismatched sequences in the central region of the miRNA target sequence, and therefore, act as non-cleavable substrates of RISC. All abovementioned inhibitors of miRNA function presumably anneale to the mature miRNA guide strand after RISC removes the passenger strand. Chemically modified oligonucleotides such as LNA and 20 -O-methylated RNA bind RNA with high affinity compared to RNA/RNA and RNA/DNA duplexes [17]. Moreover, RNA/RNA duplexes have higher Tm values than RNA/DNA duplexes, hence, in a previous study, antisense DNA did not inhibit miRNA function [9]. In this article, we reported the first effective unmodified DNA based miRNA inhibitor and showed high affinity miRNA binding to miRNA binding sites between two double-stranded regions. 2. Materials and methods 2.1. Reporter gene assay The LidNA was constructed by the heating and slow cooling of the mixture of the component oligonucleotides in PBS() buffer.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.04.013

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The resulting LidNA was analyzed by polyacrylamide gel electrophoresis. The inhibitory activity of LidNA was measured by reporter gene assay using pDsRed2-miR16 target containing three miR-16 target sequences at 30 -UTR of DsRed2 gene, using pCAGGS-GFP as the control (Fig. 1B). The HEK293T cells were seeded into 24-well plate (60 000 cells/well). At day 1, cells were transfected with the reporter and control vectors (50 ng each) and 25 nM LidNA using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. After two days, the cells were harvested and disrupted with TBS containing 0.05% of TritonX100. The mixture was centrifuged and the fluorescence of the supernatant was measured with a fluorescence microplate reader (Thermo Scientific). The inhibitory activity of LidNA was expressed as normalized DsRed2/GFP ratio relative to the control. 2.2. Binding assay of LidNA-type probe to miRNA The binding assay of LidNA-type probe to miRNA was performed on a Biacore 3000 system (GE Healthcare). On the sensor chip CM5, streptavidin was immobilized and then 50 biotin-labeled anchor oligonucleotide, 50 -tttttttttcaatacgactcactatagggc-30 , was immobilized according to the manufacturer’s instructions. LidNA-type, semi-LidNA-type, and single strand probes were injected and annealed with anchor oligonucleotide. The sequences of the probes were as follows: LidNA-type probe, 50 ctggccggaacggccagCGCCAATATTTACGTGC TGCTAgccctatagtgagtcgtatta cggatcccg-30 , semi-LidNA-type probe, 50 -cggccagCGCCAATATTTACGT GCTGCTAgccctatagtgagtcgtattacggatcccg-30 , single strand probe, 50 -cggc cagCGCCAATATTTACGTGCTGCTAttttttttttgccctatagtgagtcgtattacgga tccc g-30 . Upper case letters indicate the miR-16 binding sites, and the under-

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line indicates the double-stranded regions. Using the prepared sensor chip, miR-16 at appropriate concentration in PBS() buffer was injected into the Biacore system and the sensorgram was obtained as shown in Fig. 3. Kinetic properties were calculated according to the manufacturer’s instructions. 3. Results and discussion 3.1. Design of miRNA inhibitor with DNA The binding ability of structured molecules such as antibodies is generally believed to be higher than that of unstructured molecules such as free peptides, and simple, complementary and single-stranded DNA do not inhibit miRNA activity [9]. Hence, structured DNA is also expected to have higher affinity for miRNA bound to RISC. We designed LidNA-16 as an miR-16 inhibitor, with two miR-16 binding sites between two double-stranded regions (Fig. 1A) that repress the free motion of the miRNA binding site. Mismatch of inhibitor and miRNA in RISC is most effective in the centre of the miRNA binding site [17–19]. Hence, three LidNA analogues were constructed in which the miRNA binding site was intercepted with a 4 nt random sequence at the 12th, 4th and 18th positions from the 50 end of the miRNA binding site [LidNA16(12), LidNA-16(4) and LidNA-16(18), respectively]. Reporter gene assay for miRNA inhibitory activity was performed using pDsRed2-miR-16 target vector with three entirely complementary sequences of miR-16 downstream of DsRed2 gene stop codon and pCAGGS-GFP vector as the control (Fig. 1B). Inhibitor activity was expressed as normalized ratio of DsRed2 to GFP fluorescence. The LidNA-16(12) was an effective inhibitor of miR16 function in several cell lines, of which HEK293T, HepG2, and MCF7 are known to produce miR-16 (Fig. 1C). Consistent with previous studies, LidNA-16(4) and LidNA-16(18) were less effective, suggesting that LidNA-16(12) mediates inhibition of the miR-16 on RISC, and not to free miR-16. Because LidNA-16(12) was, therefore, used as the standard (hereafter ‘LidNA-16’). LidNA-143 did not inhibit miR-16 function, indicating that LidNA-16 is a sequence-specific inhibitor (Fig. 1C). LidNA-143 specifically inhibited the function of exogenous miR-143 in HEK293T cells, little producer of miR-143 (Fig. S1). Compared with LidNA143, the addition of LidNA-16 inhibited miR-16, 4.9-, 4.7- and 3.2-times more effectively in HEK293T, HepG2 and MCF7 cells, respectively. These values are some superior to those achived using other miRNA inhibitors, LNA and 20 -O-methylated RNA, in the same cells (Fig. S2). In dose experiments, LidNA-16 showed inhibitory activity over 10 nM, while LidNA-143 did not inhibit at 25 nM (Fig. 1C). Surprisingly, single-stranded LidNA-16 (miRNA binding site between two ssDNA strands) showed no effect at 50 nM. The inhibition effect of LidNA-16 was sustained at least at 5 days and LidNA-16 was detectable in cells at day 5 (Fig. S3). Details are under investigation.

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Fig. 1. The structure and activity of LidNA. (A) LidNA-16 (12) has two miR-16 binding sites between two double stranded regions. (B) Reporter gene assay using pDsRed2-miR-16 target containing three miR-16 target sequences at 30 -UTR of DsRed2 gene and pCAGGS-GFP as the control of transfection efficiency. (C) LidNA specifically inhibited miRNA activity by dose dependent manner. LidNA-143 and ssLidNA-16 were not effective on miR-16 activity. Using LidNA-143 at 25 nM as the standard, LidNA activity was expressed as normalized DsRed2/GFP fluorescence ratio. The sequences and structures of LidNAs and miR-16 are shown in Supplementary information.

To determine the structural components necessary for the inhibitory activity of LidNA, we designed the structural variations of LidNA as shown in Fig. 2. Variations 2 and 3 are constructed with structure 1 (LidNA-16) using an additional annealed strand to keep LidNA open on one side that is annealed with one strand of right or left double strand region and get LidNA to open the one side. Due to higher mobility of miRNA binding site the opened LidNAs, 2 and 3, were not expected to demonstrate miR-16 inhibitory effects. The effects of opened LidNAs were lower but remained significant. Structure variations 4 and 5 have a

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miRNA binding sites have different sequences of the insertion and the flanking regions. Variations 6 to 11 possess a single miRNA binding site between one or two double-stranded regions at the 50 and 30 terminals. In comparison to variations 6, 7 and 8, the variation 7 has a double-stranded region at its 50 -terminal and less effective than the variation 8 that has two double-stranded regions at both the terminals, was more effective. This tendency was well preserved in comparisons of variations 9, 10 and 11. Given that ssLidNA-16 was not effective (Fig. 2), the double-stranded

single miR-16 binding site and an miR-21 or miR-302b binding site, respectively. These variations also showed moderate effects, indicating that two miRNA binding sites of LidNA were not necessary for miRNA inhibition. In addition, variations 3, 4 and 5 showed same level of effects, indicating two miRNA binding sites were not important for higher inhibitory effects. Variation 2 showed higher effect than variation 3, therefore, the inhibitory effects may be affected by the sequences of the insertion of miRNA binding sites and double-stranded regions because the two

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Fig. 2. The activities and the structures of LidNA-16 (1) and the structure variations 2–16. The sequence of the variations is shown in Supplementary information.

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Fig. 3. The binding assay of LidNA-type probe to miRNA. LidNA-type probe (A), semi-LidNA-type probe (B) and single-strand probe (C) have miRNA binding domain with two, single- and no double-stranded regions, respectively. The sensorgrams were obtained when miR-16 at indicated concentrations (50–3.125 nM) was injected using probeimmobilized sensor chip. (D) The binding properties of three probe-types.

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regions at the terminals appear to be important for miRNA binding. The significance of double-stranded regions may also be dependent on the direction, i.e. both terminals > 30 terminal > 50 terminal. To test this, smaller variations (12–16) were designed as shown in Fig. 2. These variations were constructed with one or two miRNA binding site(s) and a double-stranded region. Variations 15 and 16 were constructed with the miRNA binding site and the doublestranded region at 50 terminal and they were less effective than variations 12, 13, and 14 with miRNA binding sites and doublestranded regions at the 30 terminal. A previous report shows that hairpin-structured DNA probes bound anti-sense strands with higher affinity than liner singlestranded DNA probes [20]. In this study, we designed an experiment to measure the affinity of LidNA-type probe for miRNA using Biacore 3000 (GE Healthcare). Three types of probe were constructed and tested: LidNA-type probe with miRNA binding site between two double-stranded regions (Fig. 3A), semiLidNA-type probe with miRNA binding site between a doublestranded region and a single-stranded region (Fig. 3B), and single-strand probe with miRNA binding site between two single-stranded regions (Fig. 3C). Binding curves obtained showed significantly distinct dissociation constants, KD, for the three types of probe. The KD of LidNA-type, semi-LidNA, and single-strand probes were calculated to be 0.54 nM, 7.1 nM, and 1.0 lM, respectively (Fig. 3D). The KD value (1.0 lM) on using the single-strand probe was surprisingly higher than the concentration (25 nM) on adding LidNA to cells as an miRNA inhibitor (Fig. 1C). DNA has a very low affinity for miRNA, therefore, any DNA, except LidNA, remains to be used as an miRNA inhibitor. If there is a double-stranded region in the vicinity to miRNA binding site like in the semi-LidNA-type probe, DNA binds to miRNA with a high affinity, and it may act as an miRNA inhibitor. The KD value of LidNA-type probe (0.54 nM) suggests that DNA binds to miRNA and acts as an miRNA inhibitor at the concentration of 10–25 nM. Comparison of the binding properties of LidNA with LNA and 20 -O-methylated RNA probes show that LidNA-type probe has better affinity than LNA and 20 -O-methylated RNA (Table S1). However, detailed binding properties of LidNA were different from LNA and 20 -O-methylated RNA. LidNA show higher association rate constant, ka, than LNA and 20 -O-methylated RNA, but higher dissociation rate constant, kd (Table S1). The binding mechanism of LidNA to miRNA is very interesting and under investigation. The existence of double-stranded regions in the vicinity enhances the binding ability of the miRNA binding site for miRNA: this concept gave rise to generation of the first miRNA inhibitor constructed with unmodified DNA. Acknowledgements This research was supported in part by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) from Japan Science and Technology Agency.

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