In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei

Abstract

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Figure 1

Telomeric guanine-quadruplex molecules. (a) Schematic drawing of a parallel guanine-quadruplex from Stylonychia telomeric DNA d(G₄T₄G₄). (b) The quadruplex is stabilized by guanine quartet layers of four cyclically hydrogen-bonded guanine residues (nitrogen atoms are shaded). The Stylonychia telomeric DNA can also adopt fold-back structures as deduced in the presence of Na⁺ (c) and in the presence of K⁺ (d). The thymine residues are arranged in four-nucleotide loops spanning either the diagonal (c) or the edges (d) of the guanine-quadruplex stem (reviewed in ref. 4). Arrows indicate strand polarity.

Figure 2

Analysis of two representative scFv proteins by RIA. mRNA of Sty3 and Sty49 was translated in vitro in the presence of radioactive methionine. After incubation with the listed substrates as competitors, the translation mixture was applied to microtiter plates with immobilized parallel guanine-quadruplex. Each bar represents the average of four measurements. Competitor concentrations are given as follows: per nucleotide for salmon sperm DNA, poly[d(GC)], and poly(dT); per oligonucleotide for hairpin and d(T₄G₄) double strand; and per base triplet for triplex DNA. The background signal (binding to neutravidin) corresponding to 3–5% of the total signal was subtracted. Nonselected clones did not give a signal above the background. Soluble quadruplex in the parallel conformation abrogated binding of both scFvs Sty3 and Sty49 to the immobilized DNA. Binding was also efficiently inhibited by adding the antiparallel quadruplex conformer to Sty49, but only to a much lower extent in the case of Sty3. None of the other soluble competitors inhibited binding to a detectable degree.

Figure 3

Evidence for guanine-quadruplex in the scFv–DNA complexes. Elution profiles in all gel-filtration experiments were recorded at 280 nm for proteins (Sty3, Sty49; dashed line), 260 nm for DNA (broken line), and at both wavelengths for the complexes (solid line, 260 nm; dotted line, 280 nm). (a) The complex formed by scFv Sty3 and the parallel quadruplex adopted by four strands of d(T₄G₄T) eluted at higher apparent molecular weight than the individual components in isolation (Upper) with an equimolar mixture of scFv and guanine-quadruplex DNA. Fractions from the higher molecular weight peak were loaded on an SDS gel and stained by Coomassie blue (Inset), confirming the presence of the scFv. The eluate was analyzed by CD spectroscopy (Lower). In the region from 250 nm to 320 nm, the spectrum of the complex (solid line) is virtually superimposable on the spectrum (broken line) of four-stranded d(T₄G₄T), exhibiting a maximum at 264 nm characteristic for the parallel quadruplex conformer (23). In this region, the protein alone (dashed line) does not have a significant CD signal. (b) The analogous experiment performed with scFv Sty49 also resulted in a quantitative shift toward higher molecular weight of the DNA when mixed with equimolar amounts of protein (line coding as for scFv Sty3). SDS/PAGE (Inset) and CD spectroscopy confirmed the presence of Sty49 and parallel guanine-quadruplex DNA in this peak, with the complex showing again the characteristic CD signal of the parallel conformer (Lower). (c) scFv Sty49 was also mixed with d(G₄T₄G₄T₄G₄T₄G₄) DNA, forming an antiparallel fold-back quadruplex (labeled Antipar.). The mixture eluted in a doublet at higher apparent molecular weight than either DNA or Sty49 in isolation (Upper). The CD spectrum of the complex peaks showed a maximum at 295 nm and a minimum at 265 nm, characteristic of the antiparallel quadruplex conformation of this DNA (23).

Figure 4

Indirect immunofluorescence staining of S. lemnae nuclei. Isolated macronuclei were incubated with scFvs Sty49 or Sty3 and analyzed by fluorescence microscopy. Synchronization of Stylonychia cells (g and h) was done as described (25). Sty49 was used in a, d–g, and i; Sty3 was used in b. (Bar in a represents 10 μm.) (a) Binding of Sty49 to a macronucleus. (b) Binding of Sty3 to a macronucleus. (c) Antibody staining in the absence of scFv probe. (d) Macronuclei were digested with S1 nuclease before Sty49 binding. (e) Macronuclei were digested with Bal31 nuclease before Sty49 binding. (f) Macronuclei were incubated with 0.2 M NaOH before Sty49 binding. (g) Binding of Sty49 to a replicating macronucleus; arrow points to the replication band. (h) Hybridization of an FITC-labeled telomeric probe to a replicating macronucleus; arrow points to the replication band. (i) Binding of Sty49 to micronuclei (Upper, phase contrast; Lower, fluorescent microscopic analysis; arrows point to micronuclei). As an internal control, in some experiments a few algae were added to the nuclei (red fluorescence in a, c, and g).