U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells (2024)

FAQs

What is the U6 promoter sequence? ›

U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi. In the design and construction of viral vectors, multiple transcription units may be arranged in close proximity in a space-limited vector.

Small double-strand siRNAs are transfected into cells where the guide strand is loaded into RISC. This activated protein and nucleic acid complex can then elicit gene silencing by binding, through perfect complementarity, to a single target mRNA sequence, thereby targeting it for cleavage and degradation.

The duplex siRNAs are passed to RISC (RNA-induced silencing complex), and the complex becomes activated by unwinding of the duplex. Activated RISC complexes can regulate gene expression at many levels. Almost certainly, such complexes act by promoting RNA degradation and translational inhibition.

RNAi is an endogenous intracellular mechanism of gene expression control that recognizes the presence within a cell of a double-stranded RNA (dsRNA) species, cleaves it into shorter (21–25 nt in length) ds-oligoribonucleotide sequences, and uses these short interfering RNAs (siRNAs) to suppress the expression of ...

U6 is a type III RNA polymerase III promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi.

Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system.

A second approach to reduce off-target effects is pooling of multiple siRNAs. It is important to notice that miRNA-like off-target effects are specific to individual sequences. Thus, reducing the concentration of the applied siRNAs will also reduce miRNA-like off-target effects.

However, delivering siRNA therapeutics to target cells is a significant challenge due to the hydrophilic and negatively charged nature of siRNA molecules. This makes it difficult for them to passively diffuse through the hydrophobic lipid bilayer of the plasma membrane [36].

How long does siRNA silence a gene? Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA.

Gene knockdown can be detected as early as 4 h and could last up to 5 days, and even 7 days in some cases [15]. However, in general, 24–96 h is the ideal time periods for accessing gene knockdown and investigating functional effects of the siRNA knockdown in cell culture.

How does siRNA therapy work? ›

Our RNAi therapeutics mimic this process by delivering specially designed small interfering RNAs (siRNAs) that, as part of RISC (RNA-induced silencing complex), bind to target disease-causing mRNA and guide their destruction like a pair of molecular scissors.

Structure: The siRNA is a 21-23 nucleotide long RNA duplex with a dinucleotide 3' overhang, whereas the miRNA is a 19-25 nucleotide RNA hairpin which forms duplex by binding with each other.

siRNA are short segments of RNA in the cell cytoplasm that can bind to mRNA and prevent a protein from being made. The way that small RNAs disable mRNA and prevent protein production is called RNA interference, or RNAi.

Endogenous triggers of RNAi pathway include foreign DNA or double-stranded RNA (dsRNA) of viral origin, aberrant transcripts from repetitive sequences in the genome such as transposons, and pre-microRNA (miRNA).

Both miRNAs and siRNAs regulate gene expression by annealing to mRNA sequence elements that are partially or fully complementary.

The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression.

In addition to its catalytic role at the heart of the spliceosome, U6 snRNA is notable for undergoing extensive structural rearrangements, including unwinding and reformation of stable internal secondary structure, and for directly interacting with >25 proteins during a single round of splicing.

The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity.

U6 Promoter: Drives expression of the downstream gRNA sequence. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs. gRNA: Guide RNA compatible with the Cas9 variant being used. Terminator: Terminates transcription of the gRNA.