Direct Comparison of d-DNA and l-DNA Strand-Displacement Reactions in Living Mammalian Cells

Oligonucleotides of opposite chirality, d and l, cannot form contiguous Watson-Crick base pairs with each other.

“because oligonucleotides of opposite chirality (d versus l) are incapable of forming contiguous WC base pairs with each other8, 9”

• cites [8] sourceNot checked — no full text of the source
• cites [9] sourceSupported

Several groups have successfully used l-DNA and l-RNA biosensors in living cells.

“Towards this goal, several groups have successfully employed l-DNA and l-RNA-based biosensors in living cells, supporting the viability of the mirror-image approach.10–13”

• cites [10] sourceNot checked — no full text of the source
• cites [11] sourceNot checked — no full text of the source
• cites [12] sourcePartly supported

  The Results and Discussion section, “Application of L-DNAzymes for Intracellular Metal Ion Imaging” (page not provided), reports an L-DNAzyme biosensor detecting Cu2+ in cultured HeLa cells. This supports one example of a mirror-image biosensor used in living cells, but this paper does not establish the claim about L-RNA biosensors or several groups. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [13] sourcePartly supported

  The Results (section 3.5) report successful use of an L-DNAzyme biosensor to detect lead in river water and fetal bovine serum. The Conclusion likewise identifies water and serum as the tested samples. The paper does not report biosensing in living cells or the use of an L-RNA biosensor, so it supports only part of the attributed claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The l-DNA reporter complex showed only a negligible fluorescence increase over the same period in cells.

“In contrast, l-R showed a negligible increase in fluorescence over the same period”

Both d-R and l-R were largely resistant to degradation in DMEM containing 10% fetal bovine.

“Interestingly, both d-R and l-R were mostly resistant to degradation in cell medium (DMEM) supplemented with 10% fetal bovine (Figure S4)”

In HeLa cells, the d-DNA reporter complex without an invading strand produced a gradual rise in fluorescence that approached the maximum signal by about 10 hours.

“Following transfection of d-R into cells, a gradual increase in fluorescence was observed, which nearly reached the maximum obtainable signal after 10 hours based on an unquenched control (Figure S3).”

A scrambled-toehold input, d-INS, did not produce an appreciable fluorescent signal after photoactivation.

“Importantly, an input strand with a scrambled toehold domain (d-INS) failed to activate an appreciable fluorescent signal upon photoactivation”

With l-Out, intracellular activation of l-R was rapid, giving more than a sixfold fluorescence increase after only four hours.

“In the presence of l-Out, intracellular activation of l-R occurred rapidly, resulting in a > 6-fold enhancement in fluorescence after only 4 hours (Figure 1b,c).”

About 70% of l-reporter complexes were activated by UV treatment, based on an unquenched positive control.

“Based on an unquenched positive control reaction, ~70% of l-reporter complexes were activated in response to UV treatment (Figure S9).”

With d-Out, d-R achieved no more than about twofold fluorescence enhancement because of high background from degradation.

“In contrast, due to high background fluorescence resulting from degradation, the d-R was unable to achieve greater than ~2-fold fluorescence enhancement in the presence of d-Out.”

The activation of the d-DNA reporter in cells without an invader is due to nuclease degradation and/or non-specific dehybridization.

“As no invading strand was provided, activation of the d-R must be attributed to nuclease degradation and/or non-specific dehybridization of the complex in cells.”

Both the d-DNA and l-DNA reaction cascades operated correctly in a cell-free setting.

“The proper operation of both d-DNA and l-DNA reaction cascades was first confirmed in a cell-free setting (Figure S6).”

The intracellular environment has unique features that can destabilize d-DNA duplexes but not their l-DNA counterparts.

“suggesting that the intracellular environment contains unique features capable of destabilizing d-DNA duplexes but not their l-DNA counterparts.”

The heterochiral cascade is specific for the correct input sequence.

“confirming the heterochiral reaction cascade behaves as intended and has specificity for the correct input sequence.”

The l-DNA duplex remains intact inside cells for extended periods.

“indicating that l-DNA duplex will remain intact within cells for extended periods of time.”

l-DNA strand-displacement reactions have fast kinetics upon activation.

“and fast reaction kinetics upon activation”

l-DNA strand-displacement reactions show minimal leak despite prolonged exposure to the intracellular environment.

“Most notably, l-DNA strand-displacement reactions show minimal “leak” despite prolonged exposure to the harsh intracellular environment”

Bio-stable l-DNA reaction components can be interfaced with native DNA inside cells using heterochiral strand displacement.

“Furthermore, using “heterochiral” strand-displacement, we demonstrate that bio-stable l-DNA reaction components can be easily interfaced with native DNA inside cells.”

l-Oligonucleotides avoid off-target hybridization with abundant cellular nucleic acids.

“l-oligonucleotides avoid off-target hybridization with abundant cellular nucleic acids.”

Little strand displacement took place before transfection.

“We confirmed that minimal strand displacement occurred prior to transfection (Figure S5).”

l-DNA reactions give dramatically better signal-to-noise ratios than d-DNA counterparts.

“which together lead to dramatically improved signal-to-noise ratios compare to their d-DNA counterparts.”

UV treatment caused minimal cytotoxicity.

“We note that minimal cytotoxicity was observed following UV treatment (Figure S8).”

A majority of the reaction components co-localized and reacted in cells even though they were transfected separately.

“This suggested that a majority of the reaction components were able to co-localize and react in cells despite being transfected separately.”

l-DNA-based strand-displacement circuits have dramatically improved intracellular performance and reliability compared with native d-DNA counterparts.

“demonstrate unequivocally that l-DNA-based stand-displacement circuits have dramatically improved intracellular performance and reliability compared to their native counterparts.”

The heterochiral approach provides proof-of-principle for interfacing endogenous d-nucleic acids with l-DNA circuits in live cells.

“but also provides an important proof-of-principle for interfacing endogenous d-nucleic acids with bio-orthogonal l-DNA circuits in live cells.”

The heterochiral approach removes input stability as a potential variable between the two reaction configurations.

“This “heterochiral” approach not only eliminates input stability as a potential variable between the two reaction configurations”

The study lays the foundation for interfacing endogenous d-nucleic acid signals with l-DNA circuits for in vivo applications.

“we have laid the foundation for interfacing endogenous d-nucleic acid signals (e.g. mRNAs) with robust l-DNA-based molecular circuits for practical, in vivo applications.”

Using l-oligonucleotides in strand-displacement circuits overcomes many key obstacles to using this technology in cells.

“Thus, use of l-oligonucleotides in the design and construction of strand-displacement circuits circumvents many of the key obstacles for utilizing this promising technology in cells.”

l-DNA strand-displacement systems have prolonged stability inside living cells.

“prolonged stability inside living cells.”