(H-298) A model system to detect virulent S. marcescens infection using an engineered restriction endonuclease mediated DNA strand displacement (resDSD) circuit

Thursday, October 12, 2023

2:00 PM – 3:00 PM PDT

Location: Exhibit Hall - Row H - Poster # 298

Presenting Author(s)

MV

Megan Van Meurs

Undergraduate Student Researcher
Department of Bioengineering, University of Washington, Washington, United States

Primary Investigator(s)

JN

Jeff Nivala

Research Assistant Professor
Computer Science Engineering, University of Washington, United States

Co-Author(s)

GS

Georg Seelig

Professor
University of Washington, Washington, United States

Last Author(s)

Nuttada Panpradist, PhD (she/her/hers)

Postdoctoral Fellow
Department of Global Health, University of Washington
Seattle, Washington, United States

Introduction:: Serratia marcescens is an opportunistic pathogen that can infect multiple human organs and is responsible for many healthcare-associated infections [1]. It has a mortality risk of up to 58% [2], and early diagnosis is crucial for timely treatment. S. marcescens secretes a unique restriction endonuclease [3], which has been recognized as a virulent factor and thus can be used as a diagnostic biomarker. To detect this restriction enzyme biomarker, we have designed and investigated a model system using novel restriction endonuclease mediated DNA strand displacement (resDSD), adapted from the enzyme-free DNA strand displacement (DSD) reaction [4]. In a typical DSD circuit, a DNA input “invading” strand invades a duplex DNA substrate, replacing the previous incumbent strand through branch migration to reveal a fluorescence molecule. In contrast, our resDSD circuit employs a restriction endonuclease enzyme input. Here, the toehold regions are concealed and blocked by a strand that the restriction enzyme can cleave. Once cleaved, the toehold region is exposed, allowing an invading strand to hybridize and initiate the DSD cascade. This study represents the first demonstration of the resDSD system. To validate the concept, we used commercially-available restriction endonuclease BamHI instead of S marcescens’ endonuclease.

Materials and Methods::

To design the resDSD circuit, an in-house MATLAB algorithm was utilized to generate random sequences of specific lengths, which were then combined with the BamHi recognition sequence. Binding probabilities and melting temperatures were calculated using IDT’s DNA Oligoanalyzer. The randomized sequences were optimized to minimize unwanted secondary structures and ensure melting temperatures were ≥ 5°C higher than the BamHi optimal temperature (37 °C). The circuit comprises three strands: (i) a 5’ Quencher labeled incumbent strand, (ii) a 3’ fluorophore labeled strand with a complementary region to the incumbent strand and a 5’ loop with a double-stranded region containing the BamHi restriction site, and (iii) an unlabeled invading DNA strand. Positive control (F) had only the fluorophore labled strand. The other conditions contained mixtures of 1µM fluorophore and 2µM quencher strands which were subjected to a 2-minute incubation at 95 °C, followed by a 40-minute annealing step from 95 °C to 25 °C. Negative control (FQ) had fluorophore and quencher strands without any BamHi or invading strand added. To differentiate the contribution of yield from DSD and resDSD, FQ mixture were mixed with 1.5-2.5 µM invading strands without adding BamHi (DSD control, FQI) or with 60 U BamHi enzyme (resDSD test case, FQIE). All four reaction conditions (F, FQ, FQI, and FQIE; n=4 each) were incubated at 37°C and read every minute for one hour (Excitation/Emission: 485/528, Gain: 55). ResDSD yields were calculated from FQIE signal / F signal. Noises from DSD were calculated from FQI signal / FQIE signal.

Results, Conclusions, and Discussions:: Optimization of our resDSD reaction (Fig.1) was performed using three invading strand concentrations. In all conditions, FQIE signals (i.e. resDSD reactions) were significantly higher than their respective FQI signals (i.e., DSD reactions); P-values (T-test, two-tailed, unequal variance, Holm-Bonferroni correction) were < 0.008, < 0.002, and < 0.0003 for 1.5, 2, and 2.5µM invading strand, respectively. At the highest concentration (2.5 µM) of the invading strand, we observed the highest resDSD yield of 93±0.2% (mean±SE) with 39±0.3% yield contributed from the DSD noise (FQI) after a 25-minute incubation. A longer incubation increased the yield to 99±0.04% but increased noise to 62±0.2%. This signal-to-noise ratio pattern was also observed when the medium invading strand concentration (2µM) was used, but the total resDSD yield was dropped to 79±0.6%, with 33±0.6% contributed from the DSD noise. At the low invading strand concentration (1.5µM), we only observed 35±1% resDSD yield with 58±1% of the yield from DSD noise. Despite these relatively high levels of noise contributed from DSD, we observed the significant effect of resDSD. New designs that increase the length of the toehold blocking region will increase the stability of the circuit gate and likely improve the specificity of our resDSD assay.
In conclusion, this work presents the initial development of resDSD, demonstrating our ability to detect the restriction enzyme by adaptation of traditional DSD circuits. By investigating this innovative resDSD approach, we aim to establish a reliable method for detecting S. marcescens based on its secretion of the restriction endonuclease. Such a diagnostic tool could contribute to early detection and prompt treatment of infection caused by this opportunistic pathogen in healthcare settings.

Acknowledgements (Optional): :

We thank our collaborators Dr. Alex Meeske at the University of Washington Microbiology, and Dr. Holly Rawizza at Harvard University Infectious Diseases and Brigham and Women's Hospital. We also thank the MISL Lab Members, with special thanks to Gwendolin Roote, Samantha Borje, and Jason Hoffman for equipment training and stimulating discussion. Megan van Meurs thanks the 2023 Mary Gates Research Scholarship for their personal funding. This research was supported by the 2022 UW Population Health Grant Tier II (MPI: Dr. Panpradist, Dr. Seelig, Dr. Nivala, Dr. Meeske, and Dr. Rawizza). Funders have no roles in the experimental design and interpretation of these results.

References (Optional): :

[1] Allen JL, Doidge NP, Bushell RN, Browning GF, Marenda MS. Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital. PLoS One. 2022 Mar 17;17(3):e0264848. doi: 10.1371/journal.pone.0264848. PMID: 35298517; PMCID: PMC8929579.

[2] Kim SB, Jeon YD, Kim JH, Kim JK, Ann HW, Choi H, Kim MH, Song JE, Ahn JY, Jeong SJ, Ku NS, Han SH, Choi JY, Song YG, Kim JM. Risk factors for mortality in patients with Serratia marcescens bacteremia. Yonsei Med J. 2015 Mar;56(2):348-54. doi: 10.3349/ymj.2015.56.2.348. PMID: 25683980; PMCID: PMC4329343.

[3] Michael J Benedik , Ulrich Strych, Serratia marcescens and its extracellular nuclease, FEMS Microbiology Letters, Volume 165, Issue 1, August 1998, Pages 1–13, https://doi.org/10.1111/j.1574-6968.1998.tb13120.x

[4] Zhang, K., Chen, YJ., Wilde, D. et al. A nanopore interface for higher bandwidth DNA computing. Nat Commun 13, 4904 (2022). https://doi.org/10.1038/s41467-022-32526-3