Year: 2026
Type: preprint
Abstract: Ultrashort (US) cell-free DNA (cfDNA) is a population of approximately 50-nucleotide single-stranded DNA molecules in human plasma. 1–3 Although it holds biological and diagnostic potential, US cfDNA escapes detection by conventional double-stranded library preparation methods. 1–3 Independent studies have linked US cfDNA to regulatory genomic regions and to sequences predicted to form noncanonical structures, prompting the hypothesis that higher-order DNA structure contributes to its molecular properties. This interpretation, however, has so far rested on computational prediction rather than experimental evidence. Here we test this hypothesis using computational, biophysical and biochemical approaches. In silico size-selected US cfDNA from 20 healthy donors was selectively enriched at putative quadruplex sequences (PQS) that overlap both experimentally observed quadruplex sequences and accessible chromatin of blood cells. Synthetic oligonucleotides corresponding to the most enriched of these loci adopted predominantly parallel G-quadruplex (G4) structures, as revealed by circular dichroism. Endogenous nucleic acids captured directly from pooled plasma by poly(A)-tailing and immobilization, without extraction, denaturation or annealing at any step, displayed folded G4 structures. Two orthogonal probes detected these structures: the BG4 antibody and the fluorogenic ligand N-methyl mesoporphyrin IX. Reciprocal competition with a third, chemically unrelated G4 ligand, pyridostatin, confirmed the signal. Together, these experiments provide direct experimental evidence for the presence of folded G4 structures in human blood plasma.
Institutions: University of Cologne, University Hospital Cologne, Centre National de la Recherche Scientifique, Central European Institute of Technology, École Polytechnique, Inserm, Laboratoire d'Optique et Biosciences, Central European Institute of Technology – Masaryk University, Institut Polytechnique de Paris, Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1298859290, G1773586305, G1935710221, G221412186, G2677629327, G2678520069, FOR 5504: Physiologische Ursachen und Konsequenzen der Genominstabilität, G291036901, G3503563290, G3735356012, G3850027771, SFB 1399: Mechanismen der MedikamentenEmpfindlichkeit und Resistenz beim kleinzelligen Bronchialkarzinom, G5489270881, G5652504370, G6286901469, Structure, biology and medical potential of blood plasma-derived ultra-short cell-free DNA, G7662032182, G8140083606, Czech Infrastructure for Integrative Structural Biology, Struktur, Biologie und medizinisches Potenzial der aus Blutplasma stammenden ultrakurzen zellfreien DNA, G856063357, G914607095
OpenAlex ID: W7212217803