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RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

ABSTRACT: RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of approximately 21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly specific pharmaceutical agents. Some limitations in using this technology exist because the preparation of siRNA in vitro and screening for siRNAs efficient in RNAi can be expensive and time-consuming processes. Here, we demonstrate that custom oligonucleotide arrays can be efficiently used for the preparation of defined mixtures of siRNAs for the silencing of exogenous and endogenous genes. The method is fast, inexpensive, does not require siRNA optimization and has a number of advantages over methods utilizing enzymatic preparation of siRNAs by digestion of longer dsRNAs, as well as methods based on chemical synthesis of individual siRNAs or their DNA templates.

SUBMITTER: Oleinikov AV

PROVIDER: S-EPMC1143701 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

Oleinikov Andrew V AV Zhao Jun J Gray Matthew D MD

Nucleic acids research 20050607 10

RNA interference (RNAi) is a process in which double-strand RNA (dsRNA) directs the specific degradation of a corresponding target mRNA. The mediators of this process are small dsRNAs, of approximately 21 bp in length, called small interfering RNAs (siRNAs). siRNAs, which can be prepared in vitro in a number of ways and then transfected into cells, can direct the degradation of corresponding mRNAs inside these cells. Hence, siRNAs represent a powerful tool for studying gene functions, as well as ...[more]

PMID: 15942024

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Project description:During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

Project description:Our goal was to identify spatial filtering methods that would improve decoding of continuous arm movements from epidural field potentials as well as demonstrate the use of the epidural signals in a closed-loop brain-machine interface (BMI) system in monkeys.Eleven spatial filtering options were compared offline using field potentials collected from 64-channel high-density epidural arrays in monkeys. Arrays were placed over arm/hand motor cortex in which intracortical microelectrodes had previously been implanted and removed leaving focal cortical damage but no lasting motor deficits. Spatial filters tested included: no filtering, common average referencing (CAR), principle component analysis, and eight novel modifications of the common spatial pattern (CSP) algorithm. The spatial filtering method and decoder combination that performed the best offline was then used online where monkeys controlled cursor velocity using continuous wrist position decoded from epidural field potentials in real time.Optimized CSP methods improved continuous wrist position decoding accuracy by 69% over CAR and by 80% compared to no filtering. Kalman decoders performed better than linear regression decoders and benefitted from including more spatially-filtered signals but not from pre-smoothing the calculated power spectra. Conversely, linear regression decoders required fewer spatially-filtered signals and were improved by pre-smoothing the power values. The 'position-to-velocity' transformation used during online control enabled the animals to generate smooth closed-loop movement trajectories using the somewhat limited position information available in the epidural signals. The monkeys' online performance significantly improved across days of closed-loop training.Most published BMI studies that use electrocorticographic signals to decode continuous limb movements either use no spatial filtering or CAR. This study suggests a substantial improvement in decoding accuracy could be attained by using our new version of the CSP algorithm that extends the traditional CSP method for use with continuous limb movement data.

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RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

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RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays.

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