Selection of DNA aptamers with two modified bases

Abstract

The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

Keywords: PCSK9; PSMA; SELEX; SOMAmer; modified aptamer.

Fig. 1.

Selection of nucleic acid ligands from DNA libraries containing C5-position modified cytidine (mod-dC) and uridine (mod-dU) nucleotides. (A) Schematic of four bases in a DNA showing double modifications with modified derivatives dC and dU. Modifications on dC are represented by the R₁ group and modifications on dU are represented by the R₂ group. (B) Chemical structures of mod-dC and mod-dU triphosphates bearing a 5-(N-substituted-carboxamide) functional group R₁ and R₂, respectively, and space-filling models of R groups as follows: Nap, 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxy; Pp, 5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxy; Moe, 5-[N-(1-morpholino-2-ethyl)carboxamide]-2′-deoxy; Tyr, 5-[N-(4-hydroxyphenyl-2-ethyl)carboxamide]-2′-deoxy; and Thr, 5-[N-(S-2-hydroxypropyl)carboxamide]-2′-deoxy. R₁ groups tested on dC were Nap and Pp (red lines), whereas R₂ groups tested on dU were Nap, Pp, Moe, Tyr, and Thr (blue lines). (C) Primer extension yields (mean ± SD, n = 3) of single-modified and double-modified libraries relative to unmodified DNA control library. Eighteen libraries were compared (SI Appendix, Fig. S1) including unmodified DNA control, single modifications on either dC (Nap and Pp) or dU (Nap, Pp, Moe, Tyr, and Thr), and all combinations of double modifications on dC and dU. (D) Nucleotide frequencies (blue bars, dC/mod-dC; red bars, dT/mod-dU; green bars, dG; and gray bars, dA) in unmodified control library, single-modified and double-modified libraries calculated from deep sequencing data from over 11,000 sequences (SI Appendix, Table S2) for each library before selection (Top) and after six rounds of affinity selection (Bottom). The ratio of nucleotides in the synthetic random template library was designed as 1:1:1:1 or 25% each.

Fig. 2.

Binding properties of 40-mer and 30-mer ligands from single-modified and double-modified libraries. (A) Binding affinities of partially truncated 40-mer SOMAmers (30 nucleotides from the originally randomized region, plus 5 nucleotides from each primer end) to PCSK9 selected from various libraries. SOMAmers with K_d ≤ 1 nM are highlighted in the gray shaded region and ligands with K_d = 320 nM showed no detectable binding with up to 32 nM PCSK9. Median ligand K_d for each library is indicated with a black horizontal line. (B) SOMAmers with K_d ≤ 1 nM (highlighted in the gray shaded region in A) were truncated to 30 nucleotides (that is, only the initially random region), and binding affinities to PCSK9 were measured. Ligands highlighted in the gray shaded region retained equivalent binding affinity after truncation from 40-mers to 30-mers. Median ligand K_d for each library is indicated with a black horizontal line. (C) Binding characterization of selected PCSK9 SOMAmers for specificity and species cross-reactivity. Target binding specificity of high-affinity PCSK9 SOMAmers from single- and double-modified libraries to other proprotein convertases (PCs). Solution affinity measurements were carried out for high-affinity ligands (40-mers, n = 33) from single-modified and double-modified libraries. The aptamers below the dotted line at 100 nM affinity indicate no detectable binding at a 100 nM concentration of protein. (C, Inset) Species cross-reactivity of SOMAmers. The affinity of single-modified truncated 30-mer SOMAmers (K_d ≤ 1 nM) to PCSK9 from human, monkey, mouse, and rat. Horizontal bars represent median values.

Fig. 3.

Identification of sandwich pairs and PCSK9 measurements. (A) Screening of sandwich pairs in a multiplex bead-based assay (Luminex). Individual avidin-conjugated bead showing captured biotinylated primary SOMAmer binding to PCSK9 and secondary SOMAmer binding to second noncompeting epitope resulting in luminescence signal through phycoerythrin conjugate of streptavidin (SA-PE) and biotin interaction. (B) Number of sandwich pairs showing high relative signal (≥50) for each library combination. (C) Number of sandwich pairs obtained from combinations of single/single (S/S), single/double (S/D), and double/double (D/D) modified libraries. (D) Statistical differentiation of patients on statin therapy vs. no statin therapy using the SOMAmer sandwich assay. PCSK9 levels were measured in 84 plasma samples divided into two groups, a control group (n = 42) and a study group in which subjects were on atorvastatin therapy (n = 42, by self-report). Horizontal bars represent median values.

Fig. 4.

Functional characterization of selected PCSK9 SOMAmers. (A) Metabolic stability of truncated 30-mer high-affinity SOMAmers from single- and double-modified libraries. Percent full-length SOMAmer is plotted as a function of time exposed to 90% human serum at 37 °C. An unmodified dC/dT control DNA sequence was compared with single-modified and double-modified SOMAmers. (B) SOMAmer inhibitors of the PCSK9:LDL-R interaction. 26/41 SOMAmers tested showed inhibition activity, 17 with high potency (IC₅₀ < 1 nM). (C) Inhibition of PCSK9 interaction with LDL-R by SL1063. SL1063 potently inhibits the interaction of wild-type PCSK9 (IC₅₀ = 2.8 nM, green circle) and mutant PCSK9 D374Y (IC₅₀ = 35 pM, yellow triangle) with LDL-R, whereas a scrambled control ligand (SL1064) showed no inhibition of wild-type PCSK9 (red circle) or mutant PCSK9 D374Y (black triangle). (D) Inhibition of PCSK9 and recovery of LDL-R levels in wild-type HepG2 cells. Wild-type PCSK9 reduces LDL-R expression levels (blue bar) in HepG2 WT cells compared with no PCSK9 treatment (purple bar). Dose-dependent PCSK9 inhibition by SL1063 (green bars), but not SL1064 (red bars), returns LDL-R expression to levels observed in untreated HepG2 cells.