Single-stranded microRNA mimics

Abstract

miRNAs are ∼22-nt RNAs that bind to the Argonaute family of proteins and have important regulatory roles in plants and animals. Here, we show that miRNAs exhibit targeting activity in cells when delivered as single strands that are 5'-phosphorylated and that contain 2'-fluoro ribose modifications. Length preferences, chemical modification sensitivity, and genome-wide seed-based targeting all suggest that this activity is Ago-based. Activity could be enhanced by annealing of segmented passenger strands containing non-nucleic acid spacers. Furthermore, screening of randomly generated sequences identified pyrimidine rich 3' cassette sequences that increased single strand activity. These results provide an initial step in the development of single-stranded miRNA mimics for therapeutic use.

FIGURE 1.

Knockdown of miR-124 target CD164 by single-strand mimics, 24 h following transfection of HCT-116 at 2 nM and 10 nM. 2′-OH is denoted in red, 2′-fluoro in green, 2′-o-methyl in black, and 5′-phosphorylation in purple. “Duplex ctrl” is a miR-124 RNA duplex, and “Neg ctrl” is a nontargeting siRNA duplex. (A) Increased activity from single strands that are phosphorylated and fluorinated. miR-124 single strands containing different patterns of 2′-fluoro modifications, with or without 5′-phosphorylation. (B) When single-strand length is varied, maximum activity is seen for miR-124 single strands of length 19–22 nt. (C) 2′-o-methyl walkthrough of a 2′-fluoro and 5′-phosphorylated miR-124 single strand shows loss of activity when a 2′-o-methyl is placed at position 2.

FIGURE 2.

Microarray analysis shows seed-based activity from a miR-124 single strand. Microarray signature 24 h after transfection of 10 nM miR-124 21-8p into HCT116 cells, plotted as ratio of fluorescence intensity (relative to mock transfection) on the y-axis and mean fluorescence intensity for the experimental and mock samples on the x-axis. Significantly down-regulated probes (P < 1 × 10⁶) are shown in green and significantly up-regulated probes in red. Hypergeometric analysis of the hexamer and heptamer content of the down-regulated 3′ UTRs showed that the most significantly enriched heptamers and hexamers are GTGCCTT and GTGCCT, respectively.

FIGURE 3.

Activity of the fluorinated and phosphorylated miR-124 single strand is enhanced by the addition of 5′ and 3′ passenger fragments, but either fragment alone decreases activity. Dose-dependent response of CD164 mRNA in HCT-116 cells, as measured by qRT-PCR. Schematic of annealed strands depicts modified strands and passenger segments, where 2′-OH is denoted in red, 2′-fluoro is green, 2′-o-methyl is black, and 5′ phosphate is purple.

FIGURE 4.

Restoration of activity by addition of 3′ and 5′ passenger strand fragments linked by carbon spacers. HCT-116 cells were transfected at 10 nM, 4 nM, and 2 nM, and CD164 mRNA was measured by qRT-PCR. 2′-OH is depicted in red, 2′-fluoro in green, 2′-o-methyl in black, LNA in light blue, 5′ phosphate in purple, and inverted abasic in yellow. The C3-spacer (ChemGenes CLP-9908) is represented by a dash.

FIGURE 5.

Analysis of randomly selected 3′ sequences in the context of the “21-8p” modification pattern. (A) Activity of miR-124 single strands with randomized bases at positions 10 through 19. CD164 mRNA levels were measured by qRT-PCR following transfection of HCT-116 cells at 10 nM. (B) Sequence logo of miR-124 single strands with activity in the top quartile. (C) Pyrimidine-rich 3′ cassette can enhance single-strand miR-122 activity. Dose response curve of miR-122 target GYS1 to increasing amounts of the indicated oligos. (Red) miR-122 control duplex, (black) nontargeting control siRNA, (green) miR-122 sequence in the “21-8p” modification pattern, and (blue) miR-122 seed sequence fused with the “9Y_2” pyrimidine-rich 3′ cassette sequence, also in the 21-8p modification pattern.