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Therapeutic siRNA: principles, challenges, and strategies.

Gavrilov K, Saltzman WM - Yale J Biol Med (2012)

Bottom Line: The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo.An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response.This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA. kseniya.gavrilov@yale.edu

ABSTRACT
RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

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Related in: MedlinePlus

A closer look at the model for siRNA guide-strand tethering by AGO2 and target-mRNA recognition and slicing. The terminal 5’ monophosphate group of the guide strand tucks in between the MID and PIWI domains of AGO2. Meanwhile, AGO2’s PAZ domain has a hydrophobic pocket that specifically recognizes the guide-strands 3’ dinucleotide overhang. This positioning opens up siRNA guide nucleotides 2-8, the “seed region,” for base pairing with complementary target mRNA, and next base pairing at nucleotides 10-11 correctly orients the scissile phosphate between these two for cleavage by AGO2’s PIWI domain, which houses the protein’s “slicer” activity [12].
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Figure 2: A closer look at the model for siRNA guide-strand tethering by AGO2 and target-mRNA recognition and slicing. The terminal 5’ monophosphate group of the guide strand tucks in between the MID and PIWI domains of AGO2. Meanwhile, AGO2’s PAZ domain has a hydrophobic pocket that specifically recognizes the guide-strands 3’ dinucleotide overhang. This positioning opens up siRNA guide nucleotides 2-8, the “seed region,” for base pairing with complementary target mRNA, and next base pairing at nucleotides 10-11 correctly orients the scissile phosphate between these two for cleavage by AGO2’s PIWI domain, which houses the protein’s “slicer” activity [12].

Mentions: The heart of the RISC complex and principal executer of RNAi-mediated silencing is the Argonaute protein [13,14]. There are four Argonaute proteins in humans (AGO 1-4), and silencing by siRNAs is accomplished via AGO2 [13]. To bring about siRNA-mediated silencing, AGO2 must tether the guide siRNA strand, extrude the passenger strand, and then undergo several cycles of target mRNA recognition, cleavage, and release while the guide strand remains bound (Figure 1) [12]. Structural studies revealed some of the mechanisms underlying AGO2’s activity. AGO2 has three functional domains, PAZ, MID, and PIWI, of which PIWI adopts an RNase H fold and is the powerhouse behind RISC’s “slicer” activity [15]. For RISC loading, structural evidence suggests that the characteristic terminal moieties of siRNA serve anchoring functions: the 3’dinucleotide is specifically recognized by the PAZ domain of Argonaute. The overhang burrows deep into a hydrophobic pocket of the domain, where the base of the terminal nucleotide can stack with an aromatic ring of one of the numerous aromatic residues that line the pocket [16-18]. Meanwhile, the 5’ phosphate group inserts between the MID and PIWI domains, binding to a magnesium ion that itself is coordinated to the C-terminus of the protein (Figure 2) [19,21]. For guide-strand selection, thermodynamic data indicates that Argonaute selects the guide strand as the one with the less thermodynamically stable 5’ end and subsequently slices the passenger strand to encourage its ejection [20].


Therapeutic siRNA: principles, challenges, and strategies.

Gavrilov K, Saltzman WM - Yale J Biol Med (2012)

A closer look at the model for siRNA guide-strand tethering by AGO2 and target-mRNA recognition and slicing. The terminal 5’ monophosphate group of the guide strand tucks in between the MID and PIWI domains of AGO2. Meanwhile, AGO2’s PAZ domain has a hydrophobic pocket that specifically recognizes the guide-strands 3’ dinucleotide overhang. This positioning opens up siRNA guide nucleotides 2-8, the “seed region,” for base pairing with complementary target mRNA, and next base pairing at nucleotides 10-11 correctly orients the scissile phosphate between these two for cleavage by AGO2’s PIWI domain, which houses the protein’s “slicer” activity [12].
© Copyright Policy - open access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375670&req=5

Figure 2: A closer look at the model for siRNA guide-strand tethering by AGO2 and target-mRNA recognition and slicing. The terminal 5’ monophosphate group of the guide strand tucks in between the MID and PIWI domains of AGO2. Meanwhile, AGO2’s PAZ domain has a hydrophobic pocket that specifically recognizes the guide-strands 3’ dinucleotide overhang. This positioning opens up siRNA guide nucleotides 2-8, the “seed region,” for base pairing with complementary target mRNA, and next base pairing at nucleotides 10-11 correctly orients the scissile phosphate between these two for cleavage by AGO2’s PIWI domain, which houses the protein’s “slicer” activity [12].
Mentions: The heart of the RISC complex and principal executer of RNAi-mediated silencing is the Argonaute protein [13,14]. There are four Argonaute proteins in humans (AGO 1-4), and silencing by siRNAs is accomplished via AGO2 [13]. To bring about siRNA-mediated silencing, AGO2 must tether the guide siRNA strand, extrude the passenger strand, and then undergo several cycles of target mRNA recognition, cleavage, and release while the guide strand remains bound (Figure 1) [12]. Structural studies revealed some of the mechanisms underlying AGO2’s activity. AGO2 has three functional domains, PAZ, MID, and PIWI, of which PIWI adopts an RNase H fold and is the powerhouse behind RISC’s “slicer” activity [15]. For RISC loading, structural evidence suggests that the characteristic terminal moieties of siRNA serve anchoring functions: the 3’dinucleotide is specifically recognized by the PAZ domain of Argonaute. The overhang burrows deep into a hydrophobic pocket of the domain, where the base of the terminal nucleotide can stack with an aromatic ring of one of the numerous aromatic residues that line the pocket [16-18]. Meanwhile, the 5’ phosphate group inserts between the MID and PIWI domains, binding to a magnesium ion that itself is coordinated to the C-terminus of the protein (Figure 2) [19,21]. For guide-strand selection, thermodynamic data indicates that Argonaute selects the guide strand as the one with the less thermodynamically stable 5’ end and subsequently slices the passenger strand to encourage its ejection [20].

Bottom Line: The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo.An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response.This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, USA. kseniya.gavrilov@yale.edu

ABSTRACT
RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

Show MeSH
Related in: MedlinePlus