Peptide Coacervates Can Protect Sequestered Oligonucleotides from Nucleases and Release Them for Transcription and Translation

Abstract

We demonstrate that coacervates, membraneless organelles formed by liquid-liquid phase separation, sequester and protect short DNA reporters and a functional luciferase gene from enzymatic degradation by various nucleases. Associative coacervates, formed by electrostatic interactions between polyhistidine peptides and ATP, inhibit degradation very efficiently. This protection arises from strong electrostatic interactions between the peptides and oligonucleotides, limiting the enzyme access to recognition and active sites. In contrast, simple coacervates based on a sticker-and-spacer model peptide exhibited limited protection. Oligonucleotide release from associative coacervates can be triggered by external stimuli such as ionic strength or temperature increases, enabling controlled release. Using a cell-free transcription-translation system, we demonstrated that in the presence of nucleases, the associative coacervate samples protected and maintained luciferase production. The ability to protect and controllably release functional genetic material makes coacervates promising candidates for further development as biocompatible delivery vehicles and components of cell-free synthetic biology platforms.

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Schematic of the in vitro coacervate capability to protect and release oligonucleotides. (A) Formation of associative and simple coacervates. (B) DNA stability assay based on FRET. (C) Coacervates capable of sequestering DNA and protecting them from nuclease degredation. Upon perturbation of the coacervate through an external stimuli, i.e., change in ionic strength, or temperature increase, the DNA is released and can be acted upon by the enzymes. Sections of the figure were created in https://BioRender.com.

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(A) Ensemble fluorescence spectra of 250 nM dsDNA reporter in associative coacervate (H₁₂) or in buffer solution. Excitation 530 nm. The dsDNA samples were whole, while the cleaved samples consisted of dsDNA that had been previously cleaved. (B) Fluorescence microscopy image of dsDNA reporter in associative coacervate (H₁₂); 532 nm excitation and 740–750 nm detection. (C) Fluorescence microscopy image of dsDNA reporter in simple coacervate (HGLGY); 532 nm excitation and 730–740 nm detection. (D) Spectra of associative coacervate (H₁₂) or simple coacervate (HGLGY) obtained with fluorscence microscopy containing the dsDNA FRET reporter after 532 nm excitation and using 10 nm detection bins. (E) Fluorescescence of coacervates containing 200 nM EcoCy3 DNA strand. The gray is a normalized spectra of both samples before centrifugation. The blue and red lines are the supernatant of associative and simple coacervate samples after centrifugation. (F) Quantitative sequestration results of the associative and simple coacervates.

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Coacervate protection capacity. Cy5/Cy3 fluorescence emission ratio was followed over time and normalized to the buffer addition samples. Enzyme was added at t = 0. Uncertainty in scatter plots is the standard deviation of triplicates. Solid lines are n = 1 experiments. (A) Buffer (solid) compared to associative coacervate (hollow, H₁₂) with ssDNA FRET reporter and either DNase I (10 u/mL), ExoI (200 u/mL), or ExoIII (1000 u/mL). (B) Buffer (solid) compared to associative coacervate (hollow, H₁₂) with dsDNA FRET reporter and either DNase I (10 u/mL), RecBCD (500 u/mL), or EcoRI (1500 u/mL). (C) Buffer (solid) compared to simple coacervate (dashed, HGLGY) with dsDNA FRET reporter and either DNase I (5 u/mL) or RecBCD (500 u/mL).

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Coacervate release for nuclease activity observation. Cy5/Cy3 fluorescence emission ratio was followed over time. Uncertainty in scatter plots is the standard deviation of triplicates. (A) Buffer solution (solids) and associative coacervate (hollow, H₁₂) with dsDNA FRET reporter and 1500 u/ml of EcoRI enzyme. At ∼1000 s, either 540 mM Na⁺ or 20 mM Mg²⁺ was added. Buffer addition was realized as a negative control. (B) Buffer solution (solids) and associative coacervate (hollow, H₉) with dsDNA FRET reporter and 10 u/ml of DNase I enzyme. At ∼1000 s, varying concentrations of Mg²⁺ were added. (C) Percentage of FRET reporter that was cleaved from the coacervate samples at end-point of experiment as a function of perturbation applied in the presence of 10 u/ml of DNase I, except for the Na⁺ data that was in the presence of 1500 u/mL of EcoRI.

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Coacervate protection and release of the dsDNA gene sequence. (A) Representative schematic of coacervate protect and release experiment in the TXTL system. Created in biorender.com. (B) Luminescence of samples that contained the dsDNA gene for Luc9 with and without associative coacervate (H₉). The samples were challenged with 20 or 1 u/mL of DNase I or just buffer (positive control). (C) Luminescence of samples that contained dsDNA gene for Luc9 with and without simple coacervate (HGLGY). The samples were challenged with 20 u/mL of DNase I or just buffer (positive control).