The group I intron trans-integration system is composed of two plasmids, pCPIPpo (19), which carries the Physarum homing endonuclease I-PpoI, and pRSTtLSU1-ClaI (20), which carries the Tetrahymena intron TtLSU1 (Tth.L1925) flanked by yeast rDNA sequence. pRSTtLSU1-ClaI has a unique ClaI site resulting from the mutation of four nucleotides in the P1 loop, which is used for the insertion of the aptamer AptHSF-RA1 in either monomeric or dimeric form. The monomer insert was generated through bi-directional extension of an overlapping pair of oligonucleotide primers purchased from Integrated DNA Technologies. The dimeric insert was synthesized by GenScript. The sequences of these ClaI inserts are given below.

RA-1(M): 5′-ATCGATGCGGCCGCGAATTCAACTGCC TTCGGGCATCGCGATACAAAATTAAGTTGAACGCGA GTTCGCGGCCGCATCGAT-3′. RA-1(D): 5′-ATCGATGCGGCCGCGTGACGTTAATT CAACTGCCTTCGGGCATCGCGATACAAAATTAAG TTGAACGCGAGTTTTCGTCATACTCCTT GGCATCGCGATACAAAATTAAGTTGAACG CGAGTTCTTCGGAATTCAACTGCCTT GGAGGCGCGGCCGCATCGAT-3′. The ligation product was transformed into the DH5α Escherichia coli strain. Single colonies containing inserts were confirmed using polymerase chain reaction (PCR) and verified by sequencing.

The parental strains for intron homing were W303-1A (MATa; leu2-3,112; trp1-1; can1-100; ura3-1; ade2-1; his3-11,15), YPH500 (MATα; ura3-52; lys2-801_amber; ade2-101_ochre; trp1-Δ63; his3-Δ200; leu2-Δ1) and BY4741(MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0). The two plasmids for trans-integration were co-transformed into the parental strains using the LiAc/ssDNA/PEG method (21). Transformants carrying both plasmids were identified on plates with SD—Ura–His media. To induce intron homing, these transformants were cultured on SGal–Ura–His plates and subcultured several times. Samples were collected at several stages for colony PCR to monitor the progress of homing until the process was complete. The primers used in this assay have the following sequences: E1 (a.k.a. JL83, #1803–1824 of yeast 25S rDNA; downstream), 5′-TCACCCCGGAATTGGTTTATCC-3′; E2 (a.k.a. JL84, #2772–2753 of yeast 25S rDNA; upstream), 5′-CGAATGGGACCTTGAATGC-3′; I (R1), 5′-CAATTTGACGGTCTTGCC-3′. Northern blot analysis was performed according to a standard protocol (22) with the following probes end-labeled using [γ-³²P] adenosine triphosphate (ATP) (GE Healthcare) and T4 polynucleotide kinase (NEB): probe for rRNA, 5′-CGAATGGGACCTTGAATGCTAGAACGTGGAAAATGAATTCC-3′; probe for aptamer, 5′-ATCGATGCGGCCGCGAACTCGCGTTC AACTTAATTTTGTATCGCGATGCCCGAAG-3′. RNA samples were run on a 1.67% agarose gel containing 2.2 M formaldehyde in 1× MOPS buffer. Hybridization was performed at 55°C overnight. The membrane was washed with 0.1× saline-sodium citrate (SSC) containing 0.5% sodium dodecyl sulfate (SDS) at 65°C.

Yeast strains were grown continuously at 30°C on SGal–Ura–His plates. To examine their phenotypes under different conditions, strains were inoculated using several serial dilutions into the following media and cultured at the temperature indicated: SGal–Ura–His media at 22°C, SGal–Ura–His media at 30°C, SGal–Ura–His media at 37°C, SGal–Ura–His media supplemented with 5 mM CuSO₄ at 30°C, SGal–Ura–His media with 0.3 mM H₂O₂ at 30°C and SGal–Ura–His media with 1 M sorbitol at 30°C. The growth of the cell patches was compared and recorded after 3–4 days’ incubation.

Liquid yeast culture grown at 22°C in log phase (O.D. 600 = 0.5–1.0.) was split into two equal parts. The first half was subjected to HS at 39°C for 20 min, while the other half remained at 22°C as the non-HS (NHS) control. Both HS and NHS yeast cells were immediately spun down and broken open with glass beads. Total RNA was extracted using hot phenol (23). For RT, 4 pmol of each primer was hybridized with 100 ng of yeast total RNA for each 10-µl reaction. The RNA was then reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen) at 37°C for 30 min and 50°C for 30 min, followed by RNase cocktail treatment to eliminate the remaining RNA. The product was diluted 200-fold, and 2 µl of the diluted product was taken for further qPCR. The qPCR was performed according to a protocol described elsewhere (24). Serial dilutions of the input RNAs were used to plot the standard curves for each gene tested. NHS genes such as U6 and ADH1 were used as internal controls to normalize the RNA levels of the HS genes and the aptamer being tested. The sequences of the primers used are given below: SSA3F, 5′-AGGGAGGCAGAACGAGTTCAGG-3′; SSA3R, 5′-CTCCAGGACCTGCGCCGGCACC-3′; HSP82F, 5′-TCTGGGAATCCAACGCTGGTGG-3′; HSP82R, 5′-CAGAATGTCTCTTGATAACTTCC-3′; SSE2F, 5′-CAATAACTCAGTACTAGCAGTTGCC-3′; SSE2R, 5′-ACCGCCGAACTCCACTTCCACAC-3′; HSP12F, 5′-TGAAGCCAGACTCTCAAAAGTC-3′; HSP12R, 5′-CTTCTTGGTTGGGTCTTCTTCACC-3′; ADH1DNF, 5′-GGACATTGTCGGTGCTGTTCTAAAGG-3′; ADH1DNR, 5′-ATTTCTGGCAAGGTAGACAAGCCG-3′; U6F, 5′-GTTCGCGAAGTAACCCTTCGTG-3′; and U6R, 5′-GAAATAAATCTCTTTGTAAAACGG-3′.

When aptamers are used inside cells as ‘intramers’ to modulate protein function, it is important to balance the expression levels of the aptamer and the target protein to achieve the desired change in target function and cellular phenotype. Several additional issues also need to be addressed, including the rate of aptamer production, correctness of folding, stability and subcellular localization. Our expression system aims primarily at generating a high level of aptamer accumulation, so that the effect of aptamers can be easily observed and evaluated. It also ensures reasonable stability of correctly folded aptamers and retains the aptamers within the nucleus, the organelle that harbors the protein targets of our aptamers. This system comprises a set of molecular constructs that ferries the aptamer-coding DNA to a specified location in the middle of each rRNA gene. The strategy exploits unique features of the nucleolar rRNA genes to achieve the goals set above: in yeast, rRNAs are transcribed at a high level by Pol I from about 150 copies of rDNA. Since each rRNA gene contains an aptamer, the high-capacity transcription of rRNA results in the production of a large amount of aptamers as byproducts.

Utilization of the power of Pol I transcription in this scheme requires the capability of inserting a heterologous DNA fragment into all copies of the rDNA and ensuring that normal rRNA production and maturation are not affected by this insertion. These problems were solved by using a group I intron trans-integration process. In our adaptation of this system, depicted in Figure 1A, we combined components from three different species. The yeast S. cerevisiae served as the host, and harbored a group I intron, TtLSU1, from the ciliate Tetrahymena thermophila (25), and a homing endonuclease, I-PpoI, from the slime mold Physarum polycephalum (26). Homing of the group I intron into yeast rDNA was driven in trans by I-PpoI, which was part of the intron PpLSU3 (Ppo.L1925). This endonuclease recognizes a 15-bp site in each of the rDNA repeats on chromosome 12 and cleaves the DNA (27). PpLSU3 and TtLSU1 are closely related. The Tetrahymena intron was chosen because its self-splicing ribozyme is far more active then that of the Physarum intron (28), and we found that it is able to accommodate an inserted sequence and retain the ability to splice quickly and accurately (20). The aptamer-coding sequence receives a ‘piggyback ride’ as an insert in the group I intron, which integrates into these rDNA repeats at the cleaved I-PpoI site. Using this system the intron RNA accumulation can reach 2% of the total RNA (19). As depicted in Figure 1B, we have designed a construct in which the aptamer is introduced as an extension of the P1 stem of the intron. At the interface between the P1 stem and the aptamer, we used a ‘GC clamp’ (29) as a structural insulator between the intron and the aptamer, so that the splicing activity of the intron would not be compromised and the aptamer would be well exposed. Figure 1.

The indirect aptamer delivery system. (A) Trans-integration of a group I intron. The three components, the host and the two plasmids, are depicted. The arrow shows the transition of rDNA to the intron-homed state caused by I-PpoI cleavage of the rDNA and insertion of the intron-aptamer. (B) Schematic diagram of the intron-aptamer. P4–P6 and P3–P9 signify the two catalytic domains of the group I intron. The sequences shown as bases depict the ClaI sites and the GC clamp extending from the P1 stem.

To demonstrate the general utility of the method, we tested it in three yeast strains commonly used in laboratories, W303-1A, YPH500 and BY4741. To form the aptamer-coding construct, a segment of DNA coding for a minimized version of AptHSF-RA1 was inserted into the sequence of the group I intron TtLSU1. The plasmid pRSTtLSU1-RA1(M), carrying the gene for this intron-aptamer, was co-transformed into the parental yeast strains together with the plasmid pCPIPpo, which carries the homing endonuclease I-PpoI under Gal 1,10 promoter control. In this arrangement, homing of the intron was controlled by the induced expression of the homing endonuclease. Since the insertion of the intron would abolish the I-PpoI site in rDNA, the homing process proceeded until all copies of rDNA had acquired an intron.

To observe this process and to assess our aptamer delivery system at the DNA level, we subcultured the transformants several times and performed PCR with two exon-specific primers (see Figure 2A, lanes labeled EE) or an exon-specific primer paired with an intron-specific primer (lanes labeled EI). Intron homing would result in increased size of the EE fragment (from 870 to 1425 bp). The 745 bp EI fragment should only be produced by rDNA with a homed intron. In Figure 2A, we show the results of one completely homed clone compared to one parental clone. Although the data shown were generated using the W303-1A parental strain, this experiment was performed in all three genetic backgrounds with identical results. Figure 2.

Functionality of the aptamer delivery system. (A) Intron homing to rDNA as confirmed by PCR. The primer annealing sites on rDNA before and after homing is given at the top. The homing of the intron to all rDNA is shown for one clone in the W303-1A parental strain. EE designates bands produced by the two exonic primers. EI designates bands produced by the exonic primer E1 and the intronic primer I. ‘M’ indicates molecular weight markers. (B) RNA expression as monitored by Northern blot analysis. ‘P’ indicates the parental strain. ‘H’ indicates the homed strain. W303-1A was used for this experiment.

The minimized ‘Core’ of the AptHSF-RA1 comprises a three-way junction. Of the three stems connected to the junction, Stem 1 contains an apical loop and an internal loop, which are required for the aptamer’s activity. Stems 2 and 3 are short and both can serve as a point of integration with other structural or functional elements. As shown in Figure 3A, in the TtLSU1-RA1(M) construct, we connected Stem 3 with the P1 stem of the intron and grafted a UUCG tetra-loop to the tip of Stem 2 to maintain strand continuity, as this arrangement was closer to the original full length version. Having successfully tested the expression system using the monomeric construct, we also created a dimeric construct. To ensure correct folding, an additional three-way junction was used to join the two aptamers and the intron together. This three-way junction was previously shown to be stable among other variants (30) and used to present multiple aptamers in molecular composites (31). As shown in Figure 3A, we used either Stem 3 or 2 to connect the aptamer to this additional three-way junction to form the dimer RA1(D). This longer and more complex sequence also helped demonstrate the general utility of the aptamer expression system. Figure 3.

Cellular phenotype of the inhibitory HSF aptamer. (A) Predicted secondary structure of the monomer and the dimer being expressed in yeast. The sequence between the two ClaI sites is shown. Different stems are indicated. The additional three-way junction used to present the dimer is encircled. (B) Growth of the monomer (M)-expressing strain and control strains cultured on different solid media and under different temperatures. ‘Antisense (R1)’ is a strain in which the antisense sequence of the monomer was expressed. (C) Growth of the dimer (D)-expressing strain and control strains cultured under different temperatures. ‘Antisense (R2)’ is a strain in which the antisense sequence of the dimer was expressed. In both (B) and (C), the parental strain was W303-1A. Patches in both (B) and (C) were inoculated by 10-fold serial dilutions of yeast stock.

HSF1 plays a predominant role in the induction of HS genes. The temperature sensitive phenotype of the aptamer-expressing yeast strains described above led us to predict that the HSF aptamer interfered with HSF activation of at least some HS genes. To investigate the mechanism underlying the systemic effect of the aptamer, we examined the mRNA level of three representative HS genes before and after heat treatment using W303-1A stains expressing the AptHSF in either monomeric (M) or dimeric (D) forms, and compared them with the strain harboring a control antisense version (R1 or R2) of each construct. These three typical HS genes, SSA3, HSP83 and SSE2, all have HSEs in the promoter and are inducible by heat. As a control, we used the HSP12 gene, which is inducible by heat or other stresses but does not contain typical HSEs in its promoter. If the aptamer were effective as an antagonist of HSF1, we would expect that the levels of transcripts in aptamer-expressing strains would be lower after HS than those in strains expressing the antisense sequence.

As shown in Figure 4A, using RT–PCR, we observed a significant decrease in heat-induced expression of all three typical HS genes in the strain expressing the AptHSF monomer. The difference in ratios of mRNA levels under HS and NHS conditions for the aptamer and antisense strains for each gene analyzed indicated a downregulation of HSF1 activity by the aptamer. In contrast, the expression level of HSP12 was not changed under either HS or NHS conditions. To confirm this result and compare the efficacy of the monomer and dimer, we used quantitative PCR (qPCR) to measure the mRNA level of the same three HS genes in both monomer- and dimer-expressing strains. Figure 4B shows a more pronounced decrease in the level of these mRNAs in the dimer-expressing strain than in the monomer-expressing strain under both HS and NHS conditions. Notably, the aptamer did not entirely inhibit HSF function, as in some cases an increase in transcription still occurred after HS. However, the resulting level of transcription apparently did not produce sufficient proteins to enable survival of the HS. In this experiment, we used the RNA polymerase II-driven ethanol-inducible gene ADH1 and the Pol III-driven constitutive gene U6 to normalize the data. These genes were not induced by HS and were not inhibited by the aptamer. Figure 4.

Molecular phenotype of the inhibitory HSF aptamer. (A) Effect of aptamer monomer (M) on the level of HS genes measured by conventional RT–PCR. ‘R1’ is a strain expressing the antisense sequence of the aptamer monomer. HS (20 min at 39°C). (B) Effect of both aptamer monomer (M) and dimer (D) on the level of HS genes measured by RT–qPCR. ‘R2’ is a strain expressing the antisense sequence of the aptamer dimer. The RNA level for each gene is presented as the ratio to the full HS induction level in the antisense control strain, which is set to 1. The expression level for each gene is normalized to that of ADH1. (We also used U6 to normalize the data, and the data sets were consistent with each other.) The error bars show the standard error from RT–qPCR experiments using three independently heat-shocked yeast RNA preparations from the same strain. In both panels the parental strain used was W303-1A.