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Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools.

Laganà A, Shasha D, Croce CM - Front Bioeng Biotechnol (2014)

Bottom Line: Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges.More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way.In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University , Columbus, OH , USA.

ABSTRACT
The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

No MeSH data available.


Artificial RNA constructs for miRNA and gene regulation. (A) Standard double strand siRNA; the anti-sense strand is the active agent which binds the target site. (B) shRNA construct; it is produced inside the target cell from a DNA construct that has been delivered to the nucleus and it expresses the anti-sense active strand. (C) The siRNA anti-sense strand binds the target mRNA with perfect complementarity. (D) Example of an a-miR sequence targeting two different sites with partial complementarity. The seed sequence of the a-miR, highlighted in bold characters, matches perfectly the target sites. (E) The antagomiR sequence (orange) perfectly matches the sequence of the target miRNA (black). (F) The Tiny LNA sequence (orange) perfectly matches the seed sequence of the target miRNA (black). (G) miRNA sponge construct with four miRNA binding sites separated by spacers. (H) Synthetic TUD construct with two exposed miRNA binding sites. (I) Model of a CRISPR sgRNA sequence binding the target DNA region. The PAM sequence (blue) is a short DNA motif juxtaposed to the DNA complementary region. The base-pairing nucleotides of the sgRNA are shown in red, while the dCas9-binding hairpin is in green.
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Figure 1: Artificial RNA constructs for miRNA and gene regulation. (A) Standard double strand siRNA; the anti-sense strand is the active agent which binds the target site. (B) shRNA construct; it is produced inside the target cell from a DNA construct that has been delivered to the nucleus and it expresses the anti-sense active strand. (C) The siRNA anti-sense strand binds the target mRNA with perfect complementarity. (D) Example of an a-miR sequence targeting two different sites with partial complementarity. The seed sequence of the a-miR, highlighted in bold characters, matches perfectly the target sites. (E) The antagomiR sequence (orange) perfectly matches the sequence of the target miRNA (black). (F) The Tiny LNA sequence (orange) perfectly matches the seed sequence of the target miRNA (black). (G) miRNA sponge construct with four miRNA binding sites separated by spacers. (H) Synthetic TUD construct with two exposed miRNA binding sites. (I) Model of a CRISPR sgRNA sequence binding the target DNA region. The PAM sequence (blue) is a short DNA motif juxtaposed to the DNA complementary region. The base-pairing nucleotides of the sgRNA are shown in red, while the dCas9-binding hairpin is in green.

Mentions: Many studies have been conducted to determine the features associated to functional siRNAs and have allowed to establish siRNA design rules. Elbashir et al. (2001b) suggest to choose the 23-nt sequence motif AA(N19)TT as binding site, where N19 means any combination of 19 nucleotides (nt) and corresponds to the sense strand of the siRNA. The complement to AA(N19) corresponds to the anti-sense strand (Figures 1A–C).


Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools.

Laganà A, Shasha D, Croce CM - Front Bioeng Biotechnol (2014)

Artificial RNA constructs for miRNA and gene regulation. (A) Standard double strand siRNA; the anti-sense strand is the active agent which binds the target site. (B) shRNA construct; it is produced inside the target cell from a DNA construct that has been delivered to the nucleus and it expresses the anti-sense active strand. (C) The siRNA anti-sense strand binds the target mRNA with perfect complementarity. (D) Example of an a-miR sequence targeting two different sites with partial complementarity. The seed sequence of the a-miR, highlighted in bold characters, matches perfectly the target sites. (E) The antagomiR sequence (orange) perfectly matches the sequence of the target miRNA (black). (F) The Tiny LNA sequence (orange) perfectly matches the seed sequence of the target miRNA (black). (G) miRNA sponge construct with four miRNA binding sites separated by spacers. (H) Synthetic TUD construct with two exposed miRNA binding sites. (I) Model of a CRISPR sgRNA sequence binding the target DNA region. The PAM sequence (blue) is a short DNA motif juxtaposed to the DNA complementary region. The base-pairing nucleotides of the sgRNA are shown in red, while the dCas9-binding hairpin is in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263176&req=5

Figure 1: Artificial RNA constructs for miRNA and gene regulation. (A) Standard double strand siRNA; the anti-sense strand is the active agent which binds the target site. (B) shRNA construct; it is produced inside the target cell from a DNA construct that has been delivered to the nucleus and it expresses the anti-sense active strand. (C) The siRNA anti-sense strand binds the target mRNA with perfect complementarity. (D) Example of an a-miR sequence targeting two different sites with partial complementarity. The seed sequence of the a-miR, highlighted in bold characters, matches perfectly the target sites. (E) The antagomiR sequence (orange) perfectly matches the sequence of the target miRNA (black). (F) The Tiny LNA sequence (orange) perfectly matches the seed sequence of the target miRNA (black). (G) miRNA sponge construct with four miRNA binding sites separated by spacers. (H) Synthetic TUD construct with two exposed miRNA binding sites. (I) Model of a CRISPR sgRNA sequence binding the target DNA region. The PAM sequence (blue) is a short DNA motif juxtaposed to the DNA complementary region. The base-pairing nucleotides of the sgRNA are shown in red, while the dCas9-binding hairpin is in green.
Mentions: Many studies have been conducted to determine the features associated to functional siRNAs and have allowed to establish siRNA design rules. Elbashir et al. (2001b) suggest to choose the 23-nt sequence motif AA(N19)TT as binding site, where N19 means any combination of 19 nucleotides (nt) and corresponds to the sense strand of the siRNA. The complement to AA(N19) corresponds to the anti-sense strand (Figures 1A–C).

Bottom Line: Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges.More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way.In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University , Columbus, OH , USA.

ABSTRACT
The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

No MeSH data available.