Design and cloning strategies for constructing shRNA expression vectors

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To address these shortcomings, we tested a panel of molecular disruption agents to reduce secondary structure formation during chain elongation. Agents tested included; Q-solution (1?, Qiagen), betaine (1 M), ammonium sulfate (AMSO, 15 mM), dimethyl sulfoxide (DMSO, 5 %) and GC-Melt (1 M, BD Biosciences). None of the additives tested yielded a detectable full-length extension product (Fig. 2a), although the addition of AMSO did improve on the number of recombinant clones (Fig. 2b). There was, therefore, little improvement over protocols employing Taq alone.
To improve upon these results, we substituted Taq polymerase with an enzyme better able to counter the secondary structure of the hairpin template. Phi29 is an enzyme that facilitates rolling circle replication by the Bacillus subtilis phage F29 [14], and as such possesses strand displacing capabilities [15]. In addition, the supplier's comparison of fifteen available polymerases suggested that Phi29 possessed the highest displacing activity (New England Biolabs). On testing Phi29 we found it was able to copy a highly structured template oligo, yielding detectable full-length product (Fig. 2a). This resulted in higher cloning efficiencies (Fig. 2b) and a lower mutation rate (10 %) when compared to Taq (50 %) (Table 1). The mutation frequency was even lower than that reported for the annealed oligo cloning strategy (20 ? 40 %) [3]. Furthermore, with a nucleotide polymerization rate ranging from 290 nt./min. @ 4?C to 2280 nt./min. @ 30?C [16] the reaction is fast, isothermal and independent of additives. We also confirm the previous finding that oligos for primer extension need only be ordered at the minimal synthesis and purification scales (0.05 ?M, desalt) [4].
The use of another enzyme with similar properties, Vent, has also been reported [4]. However, additives (DMSO and GC-Melt) and repeated thermocycling were recommended for successful extension. Whilst valid, this technique was hampered by the occurrence of cycling-induced errors. In summary, our isothermal procedure using Phi29 retains the cost benefits of primer extension and reduces manifestations of both synthesis and polymerase-induced mutations.
During this study, we generated multiple shRNA expression constructs; all of which required sequence confirmation. Given the prevalence of mutations, this step becomes imperative as suppressive activity is dependent on homology between the siRNA guide strand and target RNA [17,18]. Unfortunately, sequencing shRNA constructs is not always straightforward [3,10-12]. We often found that the standard sequencing procedure failed, again most likely due to the inability of the polymerase to read-through the highly structured template (Fig. 3a). Neither repositioning the sequencing primer, nor the addition of molecular disruption agents to the reaction were able to overcome sequencing limitations (data not shown). Although our work with Phi29 suggests an obvious solution, it was not possible to exchange the sequencing polymerase when using automated sequencing facilities.
As an alternative, we found that inclusion of a unique restriction enzyme (RE) site within the loop sequence allows the vector to be linearised and sequenced in two separate reactions; one for the sense and one for the anti-sense (Fig. 3). Our present design incorporates a centrally located XhoI site in an 8 base loop (ACTCGAGA), but it is probable that other RE sites could also be employed. We found that the digestion could be performed directly in the sample tube destined for sequencing, with no impact on sequencing quality (see Methods for details). From our survey we also noted that although uncommon, the inclusion of an RE site within the hairpin loop was not unique (used in 8 % of cases), but its only described use was to assist in screening and selection of recombinant clones [12]. In no case was there a reported link, as we propose, between RE loop design and the benefits of dual-sequencing the digested vector.
Our design incorporates an additional mismatched nucleotide pair placed adjacent to the end of the stem (ACUCGAGA, mismatches indicated in bold). Structural predictions reveal this to be a necessary inclusion to ensure that the loop, based on a palindromic RE site, remains in an open configuration (Fig. 4). This is important as additional paired nucleotides at the base of the loop effectively increase stem length, shifting the intended stem-loop junction. It has been demonstrated, for analogous microRNA structures, that altering the stem-loop junction has possible consequences for ensuing cleavage, processing, target recognition and hence suppressive activity [19] ? an observation that we have also noted for shRNA molecules (manuscript in preparation). Surprisingly, 60 % of surveyed studies employed the loop sequence, UUCAAGAGA, which is predicted to internally pair (UU.. to ..GA), potentially altering suppressive activity as described (Fig. 4). The loop design we propose is amenable to any hairpin sequence without altering the internal stem, stem-loop junction or consequent siRNA characteristics.
Another reported strategy to alleviate sequencing difficulties is to include mismatched bases within the shRNA stem [3,11]. Additionally, it has been proposed that this also reduces the occurrence of bacterially-derived mutation events. The mismatches are positioned such that the anti-sense stem (designed to be the siRNA 'guide' strand) is complementary to the target but mismatched to the sense stem (suggested as 3 or 4 'C to U', or 'A to G' conversions). We attempted this using the annealed oligo strategy yet still observed an ~27 % mutation rate ? a figure comparable to fully complementary stem designs [3]. While we did see a reduction in sequencing difficulties when mismatches were present, we also observed a correlation between increasing mismatches and decreasing gene suppression activity (Fig. 5). We can only speculate that these disparities with the original observations were due to sequence-specific effects (resulting in activity differences) or different bacterial lab strains (resulting in mutation differences). With reference to the latter, commonly used E.coli strains such as DH5a encode sbcC and sbcD, which are proteins known to generate double-stranded breaks in DNA hairpins [20]. We have found that engineered sbcCD deletion strains such as GT116 (Invivogen), specifically developed to tolerate inverted repeat regions in DNA, yield more faithful recombinant clones.
It is worthy of final note that we see no obvious correlation in our data between hairpin stem length (having generated lengths from 15 ? 45 bp) and the incidence of mutations arising during cloning or problems with sequencing. In our hands they appear largely sequence dependent as we encountered long and short hairpins that were problematic on both counts.

Conclusion

We have analyzed the literature and determined that shRNA construction is frequently associated with difficulties and can be hindered by high mutation frequencies in accordance with our own observations. Our investigations to find an improved alternative led to a variation of the primer extension method using Phi29. The procedure is swift, isothermal and independent of additives making it, in our hands, the most reliable and cost effective of all the construction techniques. In addition, we present a simple and robust solution for overcoming sequencing limitations commonly encountered with shRNA vectors. This solution is based on an RE loop design, which is amenable to any shRNA without compromising its suppressive activity. These technical modifications will be of tangible benefit to researchers looking to improve their shRNA construction process.
Methods
shRNA template generation using complementary annealed oligonucleotides
Our expression vector (derived from pSILENCER-3.0H1, Ambion) contained a human H1 polymerase-III (pol-III) promoter for shRNA expression. Each shRNA insert was designed as a synthetic duplex with overhanging ends identical to those created by restriction enzyme (RE) digestion (BamHI at the 5' and HindIII at the 3') (see Additional file 2 for diagram). The coding region for each hairpin was contained within a single oligonucleotide (upper oligo: 5'-GATCC [G/A]N(19?29)ACTCGAGAN(19?29) [G/A/C]TTTTTTGGA-3') and its complementary equivalent (lower oligo: 5'-AGCTTCCAAAAAA [G/A/C]N(19?29)ACTCGAGAN(19?29) [G/A]G-3'). These ranged in size from 60 ? 100 bases (for hairpins with 19 ? 29 bp stems). Each duplex contained a transcription initiation base (if required), the shRNA encoding region (sense stem, loop sequence and anti-sense stem), a termination spacer (if required) and a pol-III termination signal consisting of a run of at least 4 'T's. The transcription initiation base was an 'A' or 'G' (required for efficient pol-III transcription initiation) and was only included if the first base of the hairpin stem was not a purine. The termination spacer was any base but 'T' and was included only if the last base of the anti-sense stem was 'T' so as to prevent premature termination via an early run of 'T's. Oligos were ordered at the minimal synthesis and purification scales (0.05 ?M and desalt, Sigma-Genosys). Each oligo was re-suspended in water (1 ? 10 ?g/?l) and 1 ?lfrom each was added to 98 ?l of annealing solution (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), heated to 100?C for 5 minutes, slowly equilibrated to room temperature and diluted up to 10,000 fold for ligation. The insert and vector were ligated, and used to transform electrocompetent GT116 E.coli (Invivogen). Positive clones were confirmed by automated sequencing using our loop digestion method.
shRNA template generation using Phi29 primer extension
Each template oligo was similar in design to the upper oligo of the annealed oligo method (5'-GCGCGGATCC[G/A]N(19?29)ACTCGAGAN(19?29)[G/A/C]TTTTTTGGAAGCTT-3') but the ends were extended to encode the