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This difference between these two methods was presumably due to differing affinities of SYBR Green II to DNA and RNA stacking bases.
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We use a simplified free-energy model, which only includes additive contributions from stacking base pairs.
In our model, we employ additional constraints to represent stacking base pairs, which is expected to improve prediction accuracy.
The standard energy model is based on the assumption that stacking base pairs and loop entropies contribute additively to the free energy of a nucleic acid secondary structure.
However, neither this nor their emission data contradict our finding that in single- and double-stranded A·T (adenine thymine-paired) DNadenine thymine-pairedy through long-liveDNAntexcitedte states in which excitation is sharedecaysthroughbases.
The enzymatic probes were also active against the majority of bases in the region and consistent with this, CV1 probing identified very few double-stranded or stacked bases.
A272* was close to the stacked bases and involved in H-bonding with A280*.
We observed no electron density in the active site cleft corresponding to stacked bases from the overhang.
We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too.
As could be expected from the paucity of regularly stacked bases, most of these were misclassified as pure proteins (Supplementary Table S1).
These observations agree with the expectation that the lack of nearby stacked bases at duplex termini would decrease stability and result in easier unstacking.
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