Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX

Abstract

We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer ('mirror-image aptamer') that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (alpha-CGRP) with an IC50 of 3 nM at 37 degrees C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with approximately 30-40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.

Figure 1

(A) Cartoon of the RNA library and the double-stranded adapters each containing a ligate and an oligonucleotide bridge, before the ligation of the primer binding sites. The library consists of a randomized region that is flanked by 4 and 6 nt long stretches of fixed sequence (green). They serve as hybridization sites for the bridging oligonucleotides of the pre-annealed double-stranded adapters. The forward ligate contains a T7 RNA polymerase promoter at its 3′ end. Reverse bridge 1 is also used as a PCR reverse primer. Two nucleotides in the reverse bridge 1 are uridines (U) which allow for primer removal under alkaline conditions. Forward bridge and reverse ligates contain a 3′ terminal 2′-3′-dideoxynucleotide (3′H) to prevent them from mispriming in the PCR. (B) Up to 50% of all run-off transcripts contain a non-templated nucleotide (N) at their 3′ ends (red). In order to ligate these species, an alternative adapter 2 was designed. It consists of a reverse ligate 2 that lacks the 5′ terminal adenosine (A) and a reverse bridge 2 that offers the universal base inosine for hybridization opposite to the additional nucleotide. Thus, the overall length of the library does not increase.

Figure 2

Flowchart of the tailored selection scheme. The amplification steps of the Tailored-SELEX process may be performed within a single tube. The process begins with the ligation of primer binding sites to those species within the nucleic acid pool that bind to a target of interest. The ligation is assisted by deoxyoligonucleotide bridges that span the ligation site. The reverse bridges also serve as cDNA and PCR primer in the subsequent reactions. The reverse strand of the PCR product is then cleaved under alkaline conditions at a predetermined cleavage site that was introduced via the reverse primer. After neutralization and an optional ethanol precipitation, the truncated reverse strand serves as a template for the in vitro transcription which is followed by RNA purification by PAGE or by DNase treatment and spin column filtration.

Figure 3

Improved ligation yield with special adapters. The autoradiogram shows the bridge-assisted ligation of primer binding sites to a ³²P-labeled in vitro transcribed RNA library (mixture of 50 and 51mer due to the partial non-templated addition of a single nucleotide) (1). While ligation to the RNA’s 5′ end with the forward adapter (plus 25 nt) was acceptable (2), ligation to the 3′ end with reverse adapter 1 only (plus 18 nt) (3) and the overall ligation yield (plus 43 nt) (4) were insufficient, because N + 1 transcripts were not ligated. By using an equimolar mixture of reverse adapters 1 and 2, the 3′ ligation efficiency (5) and the overall ligation yield (6) were markedly improved.

Figure 4

Alkaline fission of the PCR product. The reverse strand of the PCR product is truncated by alkaline fission at the ribonucleotide positions within the reverse primer. The procedure removes both the incorporated and unincorporated reverse primer that is now dispensable. (1) Ten base pair size marker, (2) PCR product before alkaline treatment, (3) PCR product after alkaline fission of the reverse strand and the unincorporated reverse primer.

Figure 5

Course of the in vitro selection. The histogram shows the course of the in vitro selection. The fraction of the RNA pool eluted from the underivatized streptavidin or neutravidin matrix (yellow bars) and from the identical matrix after capturing RNA:peptide complexes from a solution (green bars) with the indicated peptide concentration (red triangles) is shown. Starting from round 6, selection was usually performed at three different peptide concentrations. Only the data of the minimal successful peptide concentration are shown.

Figure 6

Aligned sequences from the CGRP binding RNA pool after selection round 15. The numbers indicate the individual sequence’s frequency of occurrence. The fixed sequence parts were shaded in gray. Four different conserved motifs were identified as indicated by the background colors. The blue motif was found to occur as a 24mer or in part as an 11mer if it is flanked by the magenta-colored 16 nt long split-motif. The blue motif appeared close to the 5′ end, in the middle or close to the 3′ end of the randomized region. While the green and magenta motifs were unique to the in vitro selection at 37°C, the blue and yellow motifs were also frequent in room temperature selections that were carried out in parallel (sequences not shown). The sequences within each group seem to have partly arisen from an identical ancestor sequence with Taq polymerase-induced mutations.

Figure 7

Dose–response curve for the Spiegelmer STAR-F12 and its 3′ truncated version STAR-F12-Δ43–48. IC₅₀ values are indicated by the arrows. The Spiegelmer at different concentrations was pre-incubated with 1 nM rat α-CGRP at 37°C for 1 h. Intracellular cAMP formation in SK-N-MC cells was then stimulated for 30 min at 37°C. After cell lysis, cAMP was quantified in a chemiluminescent immunoassay.