Review papers with raw data transparency

Visual Review — Englander: "Nucleic acid base pair open states by hydrogen exchange" (PNAS 2025)

Critical appraisal — strengths and limitations

• Strength: careful synthesis of decades of HX data. Englander reunites H‑T and NMR literatures, places them in Linderstrøm‑Lang/Eigen frameworks, and quantitatively compares Kop, kop, kcl across methods and molecular sizes Englander compilation
• Strength: correct use of HX theory. The paper uses Linderstrøm‑Lang (EX1/EX2) and Eigen proton‑transfer reasoning correctly to explain why imino vs amino protons report different parameters (opening‑limited vs catalysis‑limited) Linderstrom-Lang/Eigen usage
• Important caveat — soliton hypothesis remains indirect. Multi‑base open loops (soliton‑like traveling waves) fit H‑T data but are inferred, not directly observed; theoretical objections (hydrodynamic damping, solvent friction) and lack of direct structural/time‑resolved visualization remain; Englander himself notes this and calls for experimental tests (e.g., cryo‑EM, single‑molecule approaches) Soliton hypothesis (Englander)
• Method/model dependency risk. The central claim (two modes) depends on comparing measurements from different molecular systems (long polynucleotides vs short oligos) and methods with different sensitivity windows; while plausible, residual confounders include sequence composition, salt, ends/fraying, chromatin context, and differences in catalytic conditions used in HX experiments (pH, catalyst identity) which can affect EX1/EX2 crossovers.
• Data availability/transparency. The paper includes primary datasets in‑text (Table 1 and figures) but provides no external repository of raw experimental traces; that reduces reproducibility for reanalysis and for re‑fitting mechanistic models (soliton simulations) to the reported HX time courses Data availability note (Englander)

Where the claim can be falsified / direct tests

1. Direct visualization of extended open loops migrating along long duplexes with ms lifetimes (cryo‑EM ensembles, time‑resolved cryo‑EM, or high‑speed AFM) would support Englander’s soliton model; failure to detect such features under conditions where H‑T exchange reports Kop ≈10^-2 would argue against it Suggested experimental test (cryo-EM).
2. High‑resolution single‑molecule FRET or nanopore approaches on long, labeled duplexes could detect transient multi‑base opening events with ms lifetimes and ~1% occupancy; their absence under matched conditions would falsify the multi‑base dominant hypothesis.
3. Perform matched HX experiments (H‑T and modern NMR R1ρ / relaxation dispersion) on the same sequences and lengths (progressively increasing duplex length) to map the transition from single‑base to multi‑base dominated HX as size increases; if both methods converge on a single set of parameters when applied to the same molecular construct, the two‑mode model would be challenged.

Concrete suggestions to strengthen the work

• Provide raw HX time‑course traces and fits in a public repository (digital traces for H‑T stopped‑flow and NMR relaxation dispersion), enabling reanalysis and mechanistic model fitting.
• Run complementary experiments on intermediate lengths (e.g., 30–200 bp) with both H‑T and advanced NMR (R1ρ) and SMT/NOESY variants to map how Kop and kcl change with length and sequence; include controlled catalysts and ionic strength.
• Apply high‑resolution MD/metadynamics with experimental restraints (e.g., measured Kop/kcl) to test whether multi‑base loops with preserved stacking are energetically plausible and mobile on ms timescales; compare predicted HX observables from simulations to experiment.

Novel hypotheses & experiments (testable)

• Hypothesis 1 (falsifiable): Multi‑base open loops in long polynucleotides are stabilized by partial retention of stacking energy but require a local torsional untwist nucleation event; intervening sequences with stiff GC tracts will lower Kop and impede soliton mobility — test: insert GC blocks of varying lengths into long A‑tract polymers and measure Kop/kcl by H‑T and compare with single‑molecule FRET mobility of labeled loops.
• Hypothesis 2 (falsifiable): Small oligonucleotides (≤14–20 bp) cannot support soliton‑like open loops; thus, when long duplexes are cut to oligo lengths, multi‑base HX signatures disappear — test: progressive enzymatic fragmentation of long duplexes while measuring HX to find the critical length at which multi‑base signature collapses.

Confidence and closing note

The paper is a careful, theory‑aware synthesis that plausibly resolves a long‑standing methodological conflict in nucleic acid HX. Its core empirical claim (two distinct modes seen selectively by different methods) is well supported by the collated data. The soliton mechanism is an attractively unified mechanistic model but remains a hypothesis requiring direct experimental demonstration; therefore treat the soliton claim as provocative and testable, not yet proven. Final conclusion (Englander)