Specificity and functionality of microRNA inhibitors

BackgroundMicro(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.ResultsIn 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.ConclusionsThe 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.

[1]  Samir Bouasker,et al.  Structural biology: Tracing Argonaute binding , 2009, Nature.

[2]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[3]  Yi Wen Kong,et al.  How do microRNAs regulate gene expression? , 2008, Biochemical Society transactions.

[4]  Z. Paroo,et al.  ATP-dependent human RISC assembly pathways , 2010, Nature Structural &Molecular Biology.

[5]  Claes Wahlestedt,et al.  A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites , 2005, Nucleic acids research.

[6]  V. Kim,et al.  Biogenesis of small RNAs in animals , 2009, Nature Reviews Molecular Cell Biology.

[7]  Phillip D Zamore,et al.  Sequence-Specific Inhibition of Small RNA Function , 2004, PLoS biology.

[8]  P. Zamore,et al.  Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells , 2008, Nature Protocols.

[9]  L. Lim,et al.  MicroRNA targeting specificity in mammals: determinants beyond seed pairing. , 2007, Molecular cell.

[10]  Mehul M. Vora,et al.  Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology , 2008, PloS one.

[11]  Brian D. Athey,et al.  Discriminating single-base difference miRNA expressions using microarray Probe Design Guru (ProDeG) , 2008, Nucleic acids research.

[12]  H. Seitz,et al.  Structural determinants of miRNAs for RISC loading and slicer-independent unwinding , 2009, Nature Structural &Molecular Biology.

[13]  C. Hammell.  The microRNA-argonaute complex: A platform for mRNA modulation , 2008, RNA biology.

[14]  J Kolberg,et al.  A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. , 1997, Nucleic acids research.

[15]  N. Rajewsky,et al.  Silencing of microRNAs in vivo with ‘antagomirs’ , 2005, Nature.

[16]  Olivier Voinnet,et al.  Revisiting the principles of microRNA target recognition and mode of action , 2009, Nature Reviews Molecular Cell Biology.

[17]  C. Esau,et al.  Inhibition of microRNA with antisense oligonucleotides. , 2008, Methods.

[18]  S. Freier,et al.  Potent inhibition of microRNA in vivo without degradation , 2008, Nucleic acids research.

[19]  P. Zamore,et al.  Small silencing RNAs: an expanding universe , 2009, Nature Reviews Genetics.

[20]  E. Sontheimer,et al.  Origins and Mechanisms of miRNAs and siRNAs , 2009, Cell.

[21]  Rudolf Jaenisch,et al.  Targeted Deletion Reveals Essential and Overlapping Functions of the miR-17∼92 Family of miRNA Clusters , 2008, Cell.

[22]  John G Doench,et al.  Comparison of siRNA-induced off-target RNA and protein effects. , 2007, RNA.

[23]  Vladimir Benes,et al.  A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). , 2006, RNA.

[24]  R. Einspanier,et al.  miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample , 2008, BMC Molecular Biology.

[25]  J. Doudna,et al.  A three-dimensional view of the molecular machinery of RNA interference , 2009, Nature.

[26]  M. Stoffel,et al.  Specificity, duplex degradation and subcellular localization of antagomirs , 2007, Nucleic acids research.

[27]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[28]  Maria Grahn,et al.  Analysis of siRNA specificity on targets with double-nucleotide mismatches , 2008, Nucleic acids research.

[29]  T. Tuschl,et al.  Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes , 2009, Nature.

[30]  W. Filipowicz,et al.  Relief of microRNA-Mediated Translational Repression in Human Cells Subjected to Stress , 2006, Cell.

[31]  R. Russell,et al.  Principles of MicroRNA–Target Recognition , 2005, PLoS biology.

[32]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[33]  R. Ach,et al.  Direct and sensitive miRNA profiling from low-input total RNA. , 2006, RNA.

[34]  A. Silahtaroglu,et al.  Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver , 2007, Nucleic acids research.

[35]  F. Slack,et al.  Small non-coding RNAs in animal development , 2008, Nature Reviews Molecular Cell Biology.

[36]  M. Gait,et al.  miR-122 targeting with LNA/2'-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates. , 2007, RNA.

[37]  D. Leake,et al.  Double-stranded regions are essential design components of potent inhibitors of RISC function. , 2007, RNA.

[38]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[39]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[40]  G. Nuovo,et al.  Experimental validation of miRNA targets. , 2008, Methods.