On 2015 Jan 28, JAMES STIVERS commented:

In this study, Yin et al use photo-induced electron transfer diffusion-decelerated fluorescence correlation spectroscopy (PET-ddFCS) to investigate spontaneous opening of a mismatched T-G base pair DNA (Yin Y, 2014). This elegant and potentially useful method involves electron transfer between a guanine and the singlet excited state of a tetramethylrhodamine fluorophore (TMR) that is attached via a linker to the exocyclic 4-amino group of dC in the sequence context 5´-CC^TMR TCC-3´. The results are interpreted in terms of the rate of spontaneous exposure of the guanine base opposite to T in the mismatch (“base flipping”). The measured exposure rates are ~10^4-fold less than previous T-G opening rates measured by the NMR imino exchange method (Moe JG, 1992), from which the authors conclude that PET-ddFCS measures “true” base flipping rates that are relevant for mismatched detection by base flipping DNA repair enzymes (Friedman JI, 2010), and that the NMR measurements “are irrelevant to (the) significant enzymatic base flipping”.

In this work, the authors mischaracterize the implications of previous NMR DNA imino proton exchange measurements and also the general role of dynamics in specific biomolecular recognition. The authors argue that the imino exchange measurements are irrelevant to the process of enzymatic base flipping because exchange occurs during a state when the base has not yet rotated completely from the DNA base stack [see references cited in (Friedman JI, 2010)]. Using this criterion, which is narrowly centered on the amplitude of the motion, nearly all spontaneous dynamic motions of biomolecules would be deemed irrelevant for molecular recognition.

The appropriate way of viewing the problem is to ask whether damaged or mismatched DNA base pairs have kinetically competent motions that fall on the pathway of enzymatic base flipping (Friedman JI, 2010). The kinetic competence requirement is addressed by comparing base pair opening rates with the measured rates of enzymatic base flipping [(Cao C, 2006) and references therein], and the on-pathway requirement is addressed by obtaining structures of the early stages of base flipping on enzymes (Cao C, 2006). For uracil DNA glycosylase (hUNG)(Cao C, 2006), both the kinetic and structural requirements for productive dynamic motions have been rigorously met. In contrast, the base flipping rate for the T-G mismatch measured by PET-ddFCS is ~10^4-fold too slow to account for U-A base flipping by hUNG. Assuming the authors interpretation of the measured opening rates by PET-ddFCS, their rates fail the kinetic competence criterion, requiring that enzymes actively lower the kinetic barrier for large amplitude rotations of bases from the DNA. This essential role of these enzymes has never been disputed by anyone to my knowledge.

Finally, these PET-ddFCS studies would be strengthened by further controls that would help validate the author’s interpretations. First, the approach simply measures the proximity of TMR to guanine, which could occur by any plausible mechanism such as transient intercalation into a site that is induced by the increased flexibility of a mismatched duplex. Further, the DNA construct used in this work had multiple G’s near the mismatch, any of which might be involved in electron transfer. Third, the author’s interpretation assumes that the rate-limiting step is guanine exposure, but they do not consider that many opening events may occur before a productive ET complex forms. Finally, the authors should report imino proton NMR spectra of a TMR modified duplex to confirm that this non-trivial modification does not perturb the dynamic behavior.

On 2015 Jan 28, JAMES STIVERS commented:

In this study, Yin et al use photo-induced electron transfer diffusion-decelerated fluorescence correlation spectroscopy (PET-ddFCS) to investigate spontaneous opening of a mismatched T-G base pair DNA (Yin Y, 2014). This elegant and potentially useful method involves electron transfer between a guanine and the singlet excited state of a tetramethylrhodamine fluorophore (TMR) that is attached via a linker to the exocyclic 4-amino group of dC in the sequence context 5´-CC^TMR TCC-3´. The results are interpreted in terms of the rate of spontaneous exposure of the guanine base opposite to T in the mismatch (“base flipping”). The measured exposure rates are ~10^4-fold less than previous T-G opening rates measured by the NMR imino exchange method (Moe JG, 1992), from which the authors conclude that PET-ddFCS measures “true” base flipping rates that are relevant for mismatched detection by base flipping DNA repair enzymes (Friedman JI, 2010), and that the NMR measurements “are irrelevant to (the) significant enzymatic base flipping”.

In this work, the authors mischaracterize the implications of previous NMR DNA imino proton exchange measurements and also the general role of dynamics in specific biomolecular recognition. The authors argue that the imino exchange measurements are irrelevant to the process of enzymatic base flipping because exchange occurs during a state when the base has not yet rotated completely from the DNA base stack [see references cited in (Friedman JI, 2010)]. Using this criterion, which is narrowly centered on the amplitude of the motion, nearly all spontaneous dynamic motions of biomolecules would be deemed irrelevant for molecular recognition.

The appropriate way of viewing the problem is to ask whether damaged or mismatched DNA base pairs have kinetically competent motions that fall on the pathway of enzymatic base flipping (Friedman JI, 2010). The kinetic competence requirement is addressed by comparing base pair opening rates with the measured rates of enzymatic base flipping [(Cao C, 2006) and references therein], and the on-pathway requirement is addressed by obtaining structures of the early stages of base flipping on enzymes (Cao C, 2006). For uracil DNA glycosylase (hUNG)(Cao C, 2006), both the kinetic and structural requirements for productive dynamic motions have been rigorously met. In contrast, the base flipping rate for the T-G mismatch measured by PET-ddFCS is ~10^4-fold too slow to account for U-A base flipping by hUNG. Assuming the authors interpretation of the measured opening rates by PET-ddFCS, their rates fail the kinetic competence criterion, requiring that enzymes actively lower the kinetic barrier for large amplitude rotations of bases from the DNA. This essential role of these enzymes has never been disputed by anyone to my knowledge.

Finally, these PET-ddFCS studies would be strengthened by further controls that would help validate the author’s interpretations. First, the approach simply measures the proximity of TMR to guanine, which could occur by any plausible mechanism such as transient intercalation into a site that is induced by the increased flexibility of a mismatched duplex. Further, the DNA construct used in this work had multiple G’s near the mismatch, any of which might be involved in electron transfer. Third, the author’s interpretation assumes that the rate-limiting step is guanine exposure, but they do not consider that many opening events may occur before a productive ET complex forms. Finally, the authors should report imino proton NMR spectra of a TMR modified duplex to confirm that this non-trivial modification does not perturb the dynamic behavior.