Summary information and primary citation

PDB-id
9ehc
Class
DNA
Method
X-ray (1.5 Å)
Summary
Structure of short lettuce aptamer bound to to1-3peg-biotin
Reference
Kuo YA, Chen YI, Siraj N, He Y, Yang Z, Wang Y, Batchelder-Schwab EJ, Korkmaz Z, Yonas S, Nguyen TD, Hong S, Nguyen AT, Kim S, Seifi S, Fan PH, Wu Y, Liu HW, Lu Y, Ren P, Mao C, Yeh HC (2026): "Fluorogenic Aptamer Optimization on a Massively Parallel Sequencing Platform." ACS Sens. doi: 10.1021/acssensors.5c04046.
Abstract
Fluorogenic aptamers (FAPs) are increasingly important tools for cellular sensing and pathogen diagnostics. However, enhancing their performance remains a significant challenge. Here, we introduce a massively parallel approach to optimize the DNA-based FAP Lettuce using repurposed next-generation sequencing flow cells. By replacing Lettuce's cognate fluorogen, DFHBI-1T, with TO1-biotin, we achieve a 4-fold ensemble fluorescence enhancement, and a broader fluorescence lifetime modulation range from 4.7 to 6.0 ns. Through screening 8821 Lettuce variants complexed with TO1-biotin on a <i>MiSeq</i> chip, we identify the C14T mutant which exhibits an improved dissociation constant, increased quantum yield, extended fluorescence lifetime, and enhanced emission intensity. Co-crystal structures of the aptamer/fluorogen complexes reveal that π-π stacking interactions are critical for the stable coordination of TO1-biotin within Lettuce. When tested in a cellular environment, both the screening-identified C14T mutant and a structure-guided rationally designed variant, C14dU, exhibit 15% and 18% stronger fluorescence intensities, respectively, compared with canonical Lettuce. Our massive screening and molecular dynamics simulation pipeline enable efficient FAP optimization without prior structural knowledge, yielding not only improved probes for fluorescence sensing but also deeper insights into aptamer-fluorogen interactions.
G4 notes
2 G-tetrads, 1 G4 helix, 1 G4 stem, UDUD, anti-parallel:chair, 2+2, 2(+Ln+Lw+Ln)

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List of 2 G-tetrads

 1 glyco-bond=s-s- sugar=---- groove=wnwn planarity=0.707 type=other  O+ nts=4 GGGG B.DG3,B.DG40,B.DG18,B.DG13
 2 glyco-bond=-s-s sugar=---- groove=wnwn planarity=0.726 type=bowl-2 O- nts=4 GGGG B.DG4,B.DG39,B.DG19,B.DG12

List of 1 G4-helix

In DSSR, a G4-helix is defined by stacking interactions of G-tetrads, regardless of backbone connectivity, and may contain more than one G4-stem.

Helix#1, 2 G-tetrads, INTRA-molecular, with 1 stem
 1  glyco-bond=s-s- sugar=---- groove=wnwn Major-->WC O+ nts=4 GGGG B.DG3,B.DG40,B.DG18,B.DG13
 2  glyco-bond=-s-s sugar=---- groove=wnwn WC-->Major O- nts=4 GGGG B.DG4,B.DG39,B.DG19,B.DG12
  step#1  mm(<>,outward)  area=16.10 rise=3.58 twist=15.3
  strand#1 DNA glyco-bond=s- sugar=-- nts=2 GG B.DG3,B.DG4
  strand#2 DNA glyco-bond=-s sugar=-- nts=2 GG B.DG40,B.DG39
  strand#3 DNA glyco-bond=s- sugar=-- nts=2 GG B.DG18,B.DG19
  strand#4 DNA glyco-bond=-s sugar=-- nts=2 GG B.DG13,B.DG12

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1 stacking diagram 
 1  glyco-bond=s-s- sugar=---- groove=wnwn Major-->WC O+ nts=4 GGGG B.DG3,B.DG40,B.DG18,B.DG13
 2  glyco-bond=-s-s sugar=---- groove=wnwn WC-->Major O- nts=4 GGGG B.DG4,B.DG39,B.DG19,B.DG12
  step#1  mm(<>,outward)  area=16.10 rise=3.58 twist=15.3

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List of 1 G4-stem

In DSSR, a G4-stem is defined as a G4-helix with backbone connectivity. Bulges are also allowed along each of the four strands.

Stem#1, 2 G-tetrads, 3 loops, INTRA-molecular, UDUD, anti-parallel:chair, 2+2, 2(+Ln+Lw+Ln)
 1  glyco-bond=s-s- sugar=---- groove=wnwn Major-->WC O+ nts=4 GGGG B.DG3,B.DG40,B.DG18,B.DG13
 2  glyco-bond=-s-s sugar=---- groove=wnwn WC-->Major O- nts=4 GGGG B.DG4,B.DG39,B.DG19,B.DG12
  step#1  mm(<>,outward)  area=16.10 rise=3.58 twist=15.3
  strand#1  U DNA glyco-bond=s- sugar=-- nts=2 GG B.DG3,B.DG4
  strand#2  D DNA glyco-bond=-s sugar=-- nts=2 GG B.DG40,B.DG39
  strand#3  U DNA glyco-bond=s- sugar=-- nts=2 GG B.DG18,B.DG19
  strand#4  D DNA glyco-bond=-s sugar=-- nts=2 GG B.DG13,B.DG12
  loop#1 type=lateral   strands=[#1,#4] nts=7 ATGATGC B.DA5,B.DT6,B.DG7,B.DA8,B.DT9,B.DG10,B.DC11
  loop#2 type=lateral   strands=[#4,#3] nts=4 CAGT B.DC14,B.DA15,B.DG16,B.DT17
  loop#3 type=lateral   strands=[#3,#2] nts=19 GCTTCGCAGTTCCTGCGAG B.DG20,B.DC21,B.DT22,B.DT23,B.DC24,B.DG25,B.DC26,B.DA27,B.DG28,B.DT29,B.DT30,B.DC31,B.DC32,B.DT33,B.DG34,B.DC35,B.DG36,B.DA37,B.DG38

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