siRNA-optimized Modifications for Enhanced In Vivo Activity.

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo.

Single-stranded microRNA mimics.

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.

Adenosine modification may be preferred for reducing siRNA immune stimulation.

The immune stimulation induced by short interfering RNAs (siRNAs) has been reported to be quieted or abrogated by methoxy or fluoro modifications of the 2' position of the ribose sugar. However, variables such as the type of modification, nucleotide preference, and strand bias have not been systematically evaluated. Here, we report the results of a screen of several modified siRNAs via a human peripheral blood monocyte cytokine induction assay.

In vivo activity and duration of short interfering RNAs containing a synthetic 5'-phosphate.

Endogenous and exogenous short interfering RNAs (siRNAs) require a 5'-phosphate for loading into Ago2 and cleavage of the target mRNA. We applied a synthetic 5'-phosphate to siRNA guide strands to evaluate if phosphorylation in vivo is rate limiting for maximal siRNA knockdown and duration. We report, for the first time, an in vivo evaluation of siRNAs with a synthetic 5'-phosphate compared to their unphosphorylated versions.

mRNA knockdown by single strand RNA is improved by chemical modifications.

While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB).

RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated.

Genome-wide transcription and the implications for genomic organization.

Recent evidence of genome-wide transcription in several species indicates that the amount of transcription that occurs cannot be entirely accounted for by current sets of genome-wide annotations. Evidence indicates that most of both strands of the human genome might be transcribed, implying extensive overlap of transcriptional units and regulatory elements. These observations suggest that genomic architecture is not colinear, but is instead interleaved and modular, and that the same genomic sequences are multifunctional: that is, used for multiple independently regulated transcripts and as regulatory regions.

TUF love for "junk" DNA.

The widespread occurrence of noncoding (nc) RNAs--unannotated eukaryotic transcripts with reduced protein coding potential--suggests that they are functionally important. Study of ncRNAs is increasing our understanding of the organization and regulation of genomes.

RNAi and HTS: exploring cancer by systematic loss-of-function.

Cancer develops through the successive accumulation and selection of genetic and epigenetic alterations, enabling cells to survive, replicate and evade homeostatic control mechanisms such as apoptosis and antiproliferative signals. This transformation process, however, may create vulnerabilities since the accumulation of mutations can expose synthetic lethal gene interactions and oncogene-driven cellular reprogramming ('addiction'), giving rise to new therapeutic avenues. With the completion of the human genome project, it is anticipated that the identification and characterization of genetic networks that regulate cell growth, differentiation, apoptosis and transformation will be fundamental to decoding the complexity of these processes, and ultimately, cancer itself.

A tissue specific cytochrome P450 required for the structure and function of Drosophila sensory organs.

Cytochrome P450s have generally been acknowledged as broadly tuned detoxifying enzymes. However, emerging evidence argues P450s have an integral role in cell signaling and developmental processes, via their metabolism of retinoic acid, arachidonic acid, steroids, and other cellular ligands. To study the morphogenesis of Drosophila sensory organs, we examined mutants with impaired mechanosensation and discovered one, nompH, encodes the cytochrome P450 CYP303a1.