In vitrogenerated antibodies specific for telomeric guanine-quadruplex DNA react withStylonychia lemnaemacronuclei

• DOI: 10.1073/pnas.141229498
• Publication Date: 2002-07-26T14:37:36Z
• Authors: Christiane Schaff...; Imre Berger; Jan Postberg; Jozef Hanes; Hans J. Lipps; Andreas Plückthun
• Keywords: Antiparallel (mathematics); G-quadruplex; Macronucleus

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