Stability of DNA G-quadruplexes in cell lysates
Bibliographic record
Abstract
DNA G-quadruplexes are non-canonical secondary structures that can form in guanine-rich sequences. Potential G-quadruplex forming sequences have been found in telomere and gene promoter regions of the human genome, and are potential anti-cancer drug targets (1). G-quadruplexes have been reported to interact with a number of proteins and to be unfolded by various helicases (2), however their observation and characterization in vivo is rather limited. In order to investigate the stability of G-quadruplexes in a complex protein environment, we simulate cellular conditions using HeLa cell lysates. We use Förster resonance energy transfer (FRET) spectroscopy to monitor the stability of several single stranded DNA substrates containing G-quadruplex motifs and gel electrophoresis to visualize DNA substrate integrity and eventual nuclease-induced cleavage. We observe that G-quadruplex unfolding and DNA cleavage are stronger in the presence of ATP and MgCl2. Our results suggest that G-quadruplex stability in cellular lysates is affected by nuclease cleavage and G4 binding/unfolding proteins. We have previously observed that G-quadruplexes undergo transient folding in buffer solutions with low ionic strength (3). Using single molecule FRET, we plan to investigate G-quadruplex folding/unfolding dynamics in cellular extracts. (1) Rhodes, D., & Lipps, H. J. (2015). G-quadruplexes and their regulatory roles in biology. Nucleic acids research, 43(18), 8627-8637.(2) Brázda, V., Hároníková, L., Liao, J. C., & Fojta, M. (2014). DNA and RNA quadruplex-binding proteins. International journal of molecular sciences, 15(10), 17493-17517.(3) Aznauryan, M.; Sondergaard, S.; Noer, S. L.; Schiott, B.; Birkedal, V., (2016). A direct view of the complex multi-pathway folding of telomeric G-quadruplexes. Nucleic acids research, 44, 11024-11032.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".