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96 shRNAs designed for maximal coverage of HIV-1 variants.

McIntyre GJ, Groneman JL, Yu YH, Jaramillo A, Shen S, Applegate TL - Retrovirology (2009)

Bottom Line: Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position.Assay performance was improved by dividing large targets into several shorter domains.Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Australian Technology Park, Eveleigh, NSW, Australia. glen@madebyglen.com

ABSTRACT

Background: The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains.

Results: We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 - 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains.

Conclusion: In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

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Related in: MedlinePlus

Core placement and the surrounding nucleotides. The shRNAs in this study were built around a designed 19 nucleotide siRNA core. We positioned the core at the base terminus of the shRNA (the open end), and extended the stem as necessary at the loop terminus (to 20 or 21 bp). The lower strand, or anti-sense strand region, was designed to be the intended siRNA guide strand which was therefore complementary to the target. Our shRNAs were designed so that the position of the first nucleotide of the primary core (p0) corresponds with the first base pair at the base terminus. The positions of the first and last nucleotides of the flanking p-2, p-1, p+1 and p+2 positions in the expected guide strand are shown (- positions in blue, + positions in purple). We considered the sequence of these flanking bases when selecting targets and estimating core conservations as it is presently unclear if these positions are incorporated in the processed siRNA product(s). The example shown is for a 20 bp stem, so that the last nucleotide of the p+2 position is also the last nucleotide of the loop. Likewise, shRNAs with 21 bp stems, the last nucleotide of the p+2 position is the last nucleotide of the stem.
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Figure 1: Core placement and the surrounding nucleotides. The shRNAs in this study were built around a designed 19 nucleotide siRNA core. We positioned the core at the base terminus of the shRNA (the open end), and extended the stem as necessary at the loop terminus (to 20 or 21 bp). The lower strand, or anti-sense strand region, was designed to be the intended siRNA guide strand which was therefore complementary to the target. Our shRNAs were designed so that the position of the first nucleotide of the primary core (p0) corresponds with the first base pair at the base terminus. The positions of the first and last nucleotides of the flanking p-2, p-1, p+1 and p+2 positions in the expected guide strand are shown (- positions in blue, + positions in purple). We considered the sequence of these flanking bases when selecting targets and estimating core conservations as it is presently unclear if these positions are incorporated in the processed siRNA product(s). The example shown is for a 20 bp stem, so that the last nucleotide of the p+2 position is also the last nucleotide of the loop. Likewise, shRNAs with 21 bp stems, the last nucleotide of the p+2 position is the last nucleotide of the stem.

Mentions: We developed a novel shRNA design method to ensure that the processed siRNA product(s) retained their intended level of conservation irrespective of possible variations in shRNA processing (Figure 1). Each hairpin in this study was designed around a 19 bp siRNA target that we positioned at the base terminus or 'open' end of the shRNA shown to be the primary region responsible for suppressive activity [44]. We called the 19 mer siRNA target the 'primary core', and the first nucleotide of this core the 'p0' position. The 2 adjacent overlapping 19 mers 1 and 2 nucleotides upstream of the p0 position were referred to as the p-2 and p-1 positions, and the equivalent downstream ones were p+1 and p+2. By also considering the conservation of the surrounding sequences, our design ensures that even if shRNA processing shifts within 1 – 2 nucleotides from the expected p0 position, the resultant siRNA guide strand(s) will remain fully matched to the target.


96 shRNAs designed for maximal coverage of HIV-1 variants.

McIntyre GJ, Groneman JL, Yu YH, Jaramillo A, Shen S, Applegate TL - Retrovirology (2009)

Core placement and the surrounding nucleotides. The shRNAs in this study were built around a designed 19 nucleotide siRNA core. We positioned the core at the base terminus of the shRNA (the open end), and extended the stem as necessary at the loop terminus (to 20 or 21 bp). The lower strand, or anti-sense strand region, was designed to be the intended siRNA guide strand which was therefore complementary to the target. Our shRNAs were designed so that the position of the first nucleotide of the primary core (p0) corresponds with the first base pair at the base terminus. The positions of the first and last nucleotides of the flanking p-2, p-1, p+1 and p+2 positions in the expected guide strand are shown (- positions in blue, + positions in purple). We considered the sequence of these flanking bases when selecting targets and estimating core conservations as it is presently unclear if these positions are incorporated in the processed siRNA product(s). The example shown is for a 20 bp stem, so that the last nucleotide of the p+2 position is also the last nucleotide of the loop. Likewise, shRNAs with 21 bp stems, the last nucleotide of the p+2 position is the last nucleotide of the stem.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698899&req=5

Figure 1: Core placement and the surrounding nucleotides. The shRNAs in this study were built around a designed 19 nucleotide siRNA core. We positioned the core at the base terminus of the shRNA (the open end), and extended the stem as necessary at the loop terminus (to 20 or 21 bp). The lower strand, or anti-sense strand region, was designed to be the intended siRNA guide strand which was therefore complementary to the target. Our shRNAs were designed so that the position of the first nucleotide of the primary core (p0) corresponds with the first base pair at the base terminus. The positions of the first and last nucleotides of the flanking p-2, p-1, p+1 and p+2 positions in the expected guide strand are shown (- positions in blue, + positions in purple). We considered the sequence of these flanking bases when selecting targets and estimating core conservations as it is presently unclear if these positions are incorporated in the processed siRNA product(s). The example shown is for a 20 bp stem, so that the last nucleotide of the p+2 position is also the last nucleotide of the loop. Likewise, shRNAs with 21 bp stems, the last nucleotide of the p+2 position is the last nucleotide of the stem.
Mentions: We developed a novel shRNA design method to ensure that the processed siRNA product(s) retained their intended level of conservation irrespective of possible variations in shRNA processing (Figure 1). Each hairpin in this study was designed around a 19 bp siRNA target that we positioned at the base terminus or 'open' end of the shRNA shown to be the primary region responsible for suppressive activity [44]. We called the 19 mer siRNA target the 'primary core', and the first nucleotide of this core the 'p0' position. The 2 adjacent overlapping 19 mers 1 and 2 nucleotides upstream of the p0 position were referred to as the p-2 and p-1 positions, and the equivalent downstream ones were p+1 and p+2. By also considering the conservation of the surrounding sequences, our design ensures that even if shRNA processing shifts within 1 – 2 nucleotides from the expected p0 position, the resultant siRNA guide strand(s) will remain fully matched to the target.

Bottom Line: Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position.Assay performance was improved by dividing large targets into several shorter domains.Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Australian Technology Park, Eveleigh, NSW, Australia. glen@madebyglen.com

ABSTRACT

Background: The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains.

Results: We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 - 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains.

Conclusion: In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

Show MeSH
Related in: MedlinePlus