Sequence-specific inhibition of small RNA function.
ABSTRACT: Hundreds of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified from both plants and animals, yet little is known about their biochemical modes of action or biological functions. Here we report that 2'-O-methyl oligonucleotides can act as irreversible, stoichiometric inhibitors of small RNA function. We show that a 2'-O-methyl oligonucleotide complementary to an siRNA can block mRNA cleavage in Drosophila embryo lysates and HeLa cell S100 extracts and in cultured human HeLa cells. In Caenorhabditis elegans, injection of the 2'-O-methyl oligonucleotide complementary to the miRNA let-7 can induce a let-7 loss-of-function phenocopy. Using an immobilized 2'-O-methyl oligonucleotide, we show that the C. elegans Argonaute proteins ALG-1 and ALG-2, which were previously implicated in let-7 function through genetic studies, are constituents of a let-7-containing protein-RNA complex. Thus, we demonstrate that 2'-O-methyl RNA oligonucleotides can provide an efficient and straightforward way to block small RNA function in vivo and furthermore can be used to identify small RNA-associated proteins that mediate RNA silencing pathways.
Project description:Studies on hydration are important for better understanding of structure and function of nucleic acids. We compared the hydration of self-complementary DNA, RNA and 2'-O-methyl (2'-OMe) oligonucleotides GCGAAUUCGC, (UA)6 and (CG)3 using the osmotic stressing method. The number of water molecules released upon melting of oligonucleotide duplexes, Delta(n)W, was calculated from the dependence of melting temperature on water activity and the enthalpy, both measured with UV thermal melting experiments. The water activity was changed by addition of ethylene glycol, glycerol and acetamide as small organic co-solutes. The Delta(n)W was 3-4 for RNA duplexes and 2-3 for DNA and 2'-OMe duplexes. Thus, the RNA duplexes were hydrated more than the DNA and the 2'-OMe oligonucleotide duplexes by approximately one to two water molecules depending on the sequence. Consistent with previous studies, GC base pairs were hydrated more than AU pairs in RNA, whereas in DNA and 2'-OMe oligonucleotides the difference in hydration between these two base pairs was relatively small. Our data suggest that the better hydration of RNA contributes to the increased enthalpic stability of RNA duplexes compared with DNA duplexes.
Project description:Oligonucleotides composed of 2'-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5'-disulphide-linked conjugates of 3'-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48-58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48-58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.
Project description:We report the synthesis of a novel 2'-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and incorporation into a series of small steric blocking OMe oligonucleotides targeting the apical stem-loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. Binding to TAR RNA is substantially enhanced for certain single site substitutions in the centre of the oligonucleotide, and doubly substituted anti-TAR OMe 9mers or 12mers exhibit remarkably low binding constants of <0.1 nM. G-clamp-containing oligomers achieved 50% inhibition of Tat-dependent in vitro transcription at approximately 25 nM, 4-fold lower than for a TAR 12mer OMe oligonucleotide and better than found for any other oligonucleotide tested to date. Addition of one or two OMe G-clamps did not impart cellular trans-activation inhibition activity to cellularly inactive OMe oligonucleotides. Addition of an OMe G-clamp to a 12mer OMe-locked nucleic acid chimera maintained, but did not enhance, inhibition of Tat-dependent in vitro transcription and cellular trans-activation in HeLa cells. The results demonstrate clearly that an OMe G-clamp has remarkable RNA-binding enhancement ability, but that oligonucleotide effectiveness in steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone.
Project description:High-throughput pyrosequencing of endogenous small RNAs from >95% male enriched populations of alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71) worms as well as purified spermatids from fem-3(q20) adult worms. Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks. 3 samples examined. Small RNAs from alg-3(tm1155);alg-4(ok1041);fog-2(q71) males and fog-2(q71) males. Small RNAs from spermatids isolated from ferm-3(q20) worms.
Project description:Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.
Project description:Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.
Project description:Caenorhabditis elegans contains twenty-five Argonautes, of which, only ALG-1 and ALG-2 are known to interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1, and ALG-2, as well as changes in mRNA expression in alg-5, alg-1, and alg-2 mutants. Overall design: We analyzed by high-throughput sequencing miRNA-Argonaute interactions in C. elegans and their impact on mRNA levels. The data in this series includes mRNA sequencing of loss-of-function mutants, small RNA sequencing of loss-of-function mutants, and sequencing of small RNAs that co-immunoprecipitate with each Argonaute protein.
Project description:Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with most miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline where it localizes alongside the piRNA and siRNA machinery at P granules. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline.
Project description:This study analyzes miRNA association with ALG-1 and ALG-2 in different stages during larval development of C. elegans Staged animals (L1, L2, L3, L4) - the alg-2 mutant expressing tagged-ALG-2, alg-1 mutant, and wild type controls - were lysed. ALG-1 IP or ALG-2 (GFP) IP was performed using tags from all four samples at all four timepoints. Small RNAs were released from antibodies and 5' end-labelled with AlexaFluor532. Labelled RNA was hybridized to a custom microRNA microarray platform to quantify miRNA content.
Project description:Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the <i>closo</i>-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of <i>closo</i>-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of <i>closo</i>-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of <i>closo</i>-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal <i>closo</i>-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.