WO2025075709A1 - Molecular time capsules enable transcriptomic recording in live cells - Google Patents

Abstract

The present disclosure provides molecular time capsules (MTCs) that are selfassembled protein capsules for high-fidelity RNA capture and storage inside cytoplasm from living cells.

Description

MOLECULAR TIME CAPSULES ENABLE TRANSCRIPTOMIC RECORDING IN LIVE CELLS

RELATED APPLICATION

The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63/588,495 filed October 6, 2023, which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GM 124732 awarded by the National Institutes of Health and MCB 1844668 awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND

Gene expression is dynamic and shapes future cellular states, but such temporal trajectories remain challenging to map. Many important biological processes, such as cellular differentiation and disparate survivability under stress, are seeded by subpopulations of asynchronous cells with distinct transcriptional programs (1-4). Mapping these timedependent trajectories requires capturing gene expression profiles specific to the transient and founding population before a distinguishable phenotype has emerged. However, most methods to probe global gene expression necessitate immediate destruction of the cell, preventing longitudinal tracking of cellular states. Although single-cell transcriptomics provide an opportunity to dissect the population heterogeneity (5-8), they are also end-point measurements and must rely on inference methods, such as RNA velocity (9-12), to postulate temporal dynamics.

Live-cell methods for monitoring gene expression remain limited in coverage, throughput, or time-resolution. Even with advanced multicolor fluorescence microscopy, prior knowledge on marker genes selection is required to investigate expression-to-phenotype trajectories (4, 13, 14). Live cell continual RNA extraction and monitoring methods use either force microscopy (e.g., Live-Seq (15)) to achieve full genome coverage but have a limited throughput of hundreds of cells, require specialized equipment. Recent breakthroughs in transcriptome recording by DNA-editing (16-20) provide a promising avenue to dramatically increase coverage, though they currently require many hours of active recording, and capture merely dozens of events spaced across the history of a given cell lineage. Many, though not all, of these genome recording techniques are also limited to recording a handful of preselected gene targets.

SUMMARY

Methods for on-demand direct capture of live-cell transcriptome (cytoplasmic RNA), proteome (cytoplasmic proteins), and metabolome (cytoplasmic metabolites) will not only amplify existing efforts to elucidate the transitory expression changes that drive cellular differentiation, but also enable the investigation of cell physiology in difficult-to-reach conditions, such as microbes in animal guts.

To establish such a method, self-assembled protein capsules were used to encapsulate and preserve a fraction of the cytoplasm in living cells (FIG. 1A). Capsule assembly can be controlled on-demand via an inducible promoter, creating a time- stamped record that is maintained in the cell. At a later time, such as when cells become distinguishable or accessible, the capsules can be isolated via affinity purification and their content analyzed (FIG. IB). In addition to transcriptome analysis via RNA content, these “molecular time capsules” (MTCs) also can be used for proteomic or metabolomic studies.

Several criteria are required for faithful transcriptome recording by MTC. First, the captured RNA, protein and/or metabolite content must be reproducible and have minimal capture bias. Second, the retrieved RNA, protein and/or metabolite content must have minimal contamination from non-encapsulated RNAs, proteins, and metabolites. Third, the RNA, protein, and/or metabolite record must be stably preserved over time despite changes in the host transcriptome, proteome, and/or metabolome. As demonstrated herein, these criteria are met using an exemplary de novo designed protein capsule (10) (13.5-nm radius) expressed in Escherichia coli. The timing and duration of recording is controlled via an IPTG-inducible promoter. The MTCs carry histidine tags for capsule retrieval (FIG. 3).

In some aspects, the disclosure provides methods for monitoring RNA gene, protein, and/or metabolite expression in a live cell. The methods include expressing in the live cell one or more type of protein subunit that self-assemble into a protein capsule, wherein contents of the cytoplasm of the live cell are encapsulated by the self-assembled protein capsule and isolating the self-assembled protein capsule from the live cell.

In some embodiments, the self-assembled protein capsule is a synthetic nucleocapsid, evolved nucleocapsid, or synthetic viral capsid. In some embodiments, the self-assembled protein capsule has a net internal positive charge. In some embodiments, the self-assembled protein capsule has a net internal negative charge. In some embodiments, the self-assembled protein capsule comprising at least two different types of subunits.

In some embodiments, the synthetic viral capsid is the major capsid protein VP1 of the human JC virus or Ty3 GAG3.

In some embodiments, the self-assembled protein capsule comprises one or more natural, evolved, or synthetic protein organelles. In some embodiments, the protein organelle is a bacterial encapsulin or a ferritin.

In some embodiments, the self-assembled protein capsule captures RNA, protein, and/or metabolite inside the cytoplasm of the cell. In some embodiments, the self-assembled protein capsule captures RNA inside the cytoplasm of the cell. In some embodiments, the self-assembled protein capsule stores the RNA, protein, and/or metabolite inside the cytoplasm. In some embodiments, the self-assembled protein capsule stores RNA inside the cytoplasm.

In some embodiments, the methods for monitoring RNA gene, protein, and/or metabolite expression in a live cell further include isolating the RNA, protein, and/or metabolite from the isolated self-assembled protein capsule. In some embodiments, the methods for monitoring RNA gene, protein, and/or metabolite expression in a live cell further include isolating the RNA from the isolated self-assembled protein capsule.

In some embodiments, the methods for monitoring RNA gene, protein, and/or metabolite expression in a live cell further include sequencing the isolated RNA.

In some embodiments, one or more inducible promoter is operably linked to one or more sequence encoding the one or more type of protein subunit. In some embodiments, the inducible promoter is a chemically-inducible promoter, a temperature-inducible promoter, or a light-regulated promoter. In some embodiments, the chemically-inducible promoter is an isopropyl P-D-l -thiogalactopyranoside promoter (IPTG), an alcohol-regulated promoter, a tetracycline -regulated promoter, a steroid-regulated promoter, a metal-regulated promoter. In some embodiments, the inducible promoter is a tissue specific promoter, a pathogenesis- regulated promoter, a bacteriophage promoter, a bacterial promoter, or a hybrid of a bacteriophage and bacterial promoter.

In some embodiments, the timing and duration of the RNA, protein, and/or metabolite encapsulation by the self-assembled protein capsule is controlled by adding to the live cell and subsequently withdrawing from the live cell the inducer of the inducible promoter.

In some embodiments, the one or more protein subunits of the self-assembled protein capsule comprises at least one purification tag. In some embodiments, the purification tag is a histidine tag, polyarginine tag, GTS tag, FLAG tag, SBP tag, strep-tag II, calmodulin binding protein, chitin-binding tag, maltose-binding tag, or cellulose-binding tag. In some embodiments, the purification tag is a histidine tag. In some embodiments, the histidine tag is at the N-terminus of the one or more types of protein subunits. In some embodiments, the histidine tag is at the C-terminus of the one or more types of protein subunits.

In some embodiments, the self-assembled protein capsule encapsulating RNA, protein, and/or metabolite from the live cell is separated from the live cell by metal affinity chromatography, optionally by binding to a nickel chromatography resin.

In some embodiments, the cell is a prokaryotic cell or eukaryotic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1C depict the informative capture of mRNAs by genetically encoded inducible molecular time capsules (MTCs). FIG. 1A is a depiction of MTCs capable of capturing RNA in the cytoplasm. FIG. IB is a depiction of the MTCs being induced to capture a snapshot of the transcriptome. The MTCs protect the captured transcriptome for delayed retrieval and analysis. FIG. 1C shows the comparison of mRNA levels in lysate with the mRNA levels captured in the MTC.

FIGs. 2A-2E show MTCs provide stable storage with cleanly retrievable contents. FIG. 2A depicts the total lysate and MTC samples being removed from a mixed “barnyard” sample of B. subtilis and MTC-containing E. coli. FIG. 2B shows the percentage of reads in the MTC and lysate samples mapping to the E. coli and B. subtilis genome. Practically all reads in the MTC sample map to the E. coli genome - the expected outcome if the capsules robustly protect transcripts during the purification process. FIG. 2C shows the gene-by-gene scatterplot of read per million (RPM) for E. coli (x-axis) and B. subtilis (y-axis) transcripts purified from the lysate (top) and MTC capsules (bottom). The MTCs do not show any noticeable bias towards which B. subtilis RNAs they capture - achieving uniformly low coverage even amongst the most prevalent B. subtilis transcripts FIG. 2D depicts the procedure to determine how much MTCs change over time. Two time points were collected, before and after stressful treatment with 4% ethanol. FIG. 2E shows mRNA levels pre and post stress in the lysate sample and MTC sample. FIG. 3 shows protein purification of MTCs using a lysis/resuspension buffer with 150mM Imidazole and a wash buffer with 150mM Imidazole. Beads from the column were also run to demonstrate residual binding of the complex to the column.

FIG. 4 shows the growth rate of induced and uninduced wild type (WT) MG1655 E. coli and MG 1655 E. coli with MTCs (MTC). Induced samples had ImM IPTG added at back-dilution, “t” corresponds to calculated doubling time for each sample.

FIGs. 5A-5F show the reproducibility of the MTCs encapsulation of RNA per gene RPM (FIGs. 5A-5C) and general lysate RNA per gene RPM (FIGs. 5D-5F) across three samples.

FIG. 6 shows MTC encapsulated transcripts versus lysate transcripts across three biological samples.

FIG. 7A-7B shows length distribution of MTC encapsulated RNAs in comparison to general lysate RNAs. FIG. 7A shows a representative read length distribution of MTC- protected transcripts taken before the 200nt size selection step in the library preparation. FIG. 7B shows a representative read length distribution of the corresponding lysate sample transcripts, dominated by rRNA bands at ~16k nt and ~29-30k nt.

FIG. 8 shows an illustration of the plasmid map used for this work (plasmid pMP026). The I53-50-v4N genome represents the MTC transcripts, which comprises two parts: a pentameric and a trimeric subunit. The plasmid is resistant to Kanamycin. It has a pMB 1 origin, and uses a copy of lac to repress production of the MTCs.

DETAILED DESCRIPTION

Live-cell transcriptomic recording can elucidate hidden cellular states that precede phenotypic transformation. The present disclosure demonstrates the use of self-assembled protein capsules for high-fidelity RNA, protein, and/or metabolite capture and storage inside cytoplasm from living cells. Molecular time capsules (MTCs) were developed to record time- stamped transcriptome snapshots and preserve them after cellular transitions, thereby enabling retrospective investigations of gene-expression, protein-expression, and/or metabolite expression differences, such as those that drive distinct developmental outcomes.

This approach provides several key advantages over existing transcriptome recording methods. First, MTCs encapsulate RNAs in a highly reproducible manner, enabling comparative analysis of recording from different samples. Second, the recording can be completed within one hour, eliminating the need to integrate transcriptome changes over several cell generations and a much longer period of time. Thirdly, in contrast to other techniques which also encapsulate RNA in capsules, for example the recent COURIER (25) technique, which exports capsule vesicles to facilitate intercellular communication, one advantage of the MTCs is that they physically remain in the cell-lineage they were formed in. This fact of using cells to aid in the identifiability of MTC-records suggests applications where later cell-state serves as a sorting phenotype. The MTCs retrieved from sorted cells will serve as historical records of this specified sub-population. Furthermore, the MTCs can be formed in hard-to-access locations - such as in bacteria in the human gut - enabling observation of transcriptomes in situ. As the MTCs do not rely on E. coli - specific molecular machinery and makes use of a simple mechanism for capture and protection, we anticipate that this tool will prove easy to generalize to new systems, both eukaryotic and prokaryotic. Finally, the recording capacity of MTCs per cell is substantially larger than methods that utilize CRISPR spacer arrays or protein tapes.

Non-Targeted CRISPR recording systems (NTCRSs) are rate limited by the probability per unit time of an RNA being reverse transcribed and inserted into a CRISPR array in any given E. coli cell. Recent estimates of this number estimate that only 1 in ~1.9 x 10⁴ E. coli will acquire a spacer during a recording window of > 12 h. In contrast, the methods disclosed herein are estimated to recover the equivalent of -6.8 MTCs per cell, an estimated 3.4 x 10⁴-fold increase over NTCRSs. Further, NTCRSs can take approximately 12 hours, whereas the current methodology disclosed herein has a recording time as short as one hour. Shorter recording times lead to higher temporal resolution of the transcriptomic record, which can in turn lead to better inference about the observed transcriptomic state. The transcriptomic recording times should be on the same scale as the events they wish to capture, for example, bacteria whose RNAs have very short half-lives and which can double as frequently as every 20 minutes.

The current long recording time of NTCRSs makes it difficult to assess reproducibility. Because the transcriptome changes so much during the time of recording (-12 hours during which the cell transitions from exponential to stationary) it is to be expected that the captured record does not look like the last time point of the transcriptome, and that two biological replicates will not look alike. This issue likely explains the lack of sample-to-sample reproducibility seen in NTCRS systems (The highest Pearson Coefficient seen between biological replicates using NTCRS recording is: 0.786, much lower than the lowest Pearson Coefficient seen between biological replicates of MTC recording: 0.971(FIGs. 5A-5C)). Aspects of the present disclosure provide methods for monitoring gene expression (RNA transcription) in a live cell. In some embodiments, the method for monitoring gene expression (RNA transcription) in a live cell comprises expressing in the live cell one or more types of subunits that self-assemble into a protein capsule.

In some, aspects the present disclosure provides methods for monitoring protein expression in a live cell. In some embodiments, the method for monitoring protein expression in a live cell comprises expressing in the live cell one or more types of subunits that selfassemble into a protein capsule.

In some, aspects the present disclosure provides methods for monitoring metabolite expression in a live cell. In some embodiments, the method for monitoring metabolite expression in a live cell comprises expressing in the live cell one or more types of subunits that self-assemble into a protein capsule.

In some embodiments, the self-assembled protein capsule is an MTC. In some embodiments, the protein capsule comprises at least two different types of subunits.

In some embodiments, the self-assembled protein capsule is encoded by at least one nucleic acid sequence. In some embodiments, the self-assembled protein capsule is encoded by at least one DNA sequence. In some embodiments, the self-assembled protein capsule is encoded by at least one RNA sequence.

In some embodiments the self-assembled protein capsule encapsulates and preserves a fraction of the cytoplasm of a living cell, thereby recording and preserving the transcriptome of the cell present at the time of assembly of the protein capsule. In some embodiments, the self-assembled protein capsule encapsulates RNA inside the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule protects the encapsulated RNA from degradation in the cell, and during and after isolation of the protein capsule from the cell.

In some embodiments, the self-assembled protein capsule stores RNA. In some embodiments, the self-assembled protein capsule stores RNA encapsulated from the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule stores the encapsulated RNA inside the cytoplasm of the cell.

In some embodiments, the self-assembled protein capsule has a net internal positive charge. The net internal positive charge may facilitate retention of nucleic acids including the RNA molecules that comprise the transcriptome of the cell in which the protein capsule is assembled. The net internal positive charge is the sum of the positive charges of the protein subunits less the negative charges of the protein subunits. In other embodiments, the self- assembled protein capsule has a net internal negative charge, or a net internal charge that is substantially neutral.

In some embodiments the self-assembled protein capsule encapsulates and preserves a fraction of the cytoplasm of a living cell, thereby recording and preserving the proteome of the cell present at the time of assembly of the protein capsule. In some embodiments, the selfassembled protein capsule encapsulates protein inside the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule protects the encapsulated protein from degradation in the cell, and during and after isolation of the protein capsule from the cell.

In some embodiments, the self-assembled protein capsule stores protein. In some embodiments, the self-assembled protein capsule stores protein encapsulated from the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule stores the encapsulated protein inside the cytoplasm of the cell.

In some embodiments the self-assembled protein capsule encapsulates and preserves a fraction of the cytoplasm of a living cell, thereby recording and preserving the metabolome of the cell present at the time of assembly of the protein capsule. In some embodiments, the selfassembled protein capsule encapsulates metabolites inside the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule protects the encapsulated metabolites from degradation in the cell, and during and after isolation of the protein capsule from the cell.

In some embodiments, the self-assembled protein capsule stores metabolites. In some embodiments, the self-assembled protein capsule stores metabolites encapsulated from the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule stores the encapsulated metabolites inside the cytoplasm of the cell.

In some embodiments the self-assembled protein capsule encapsulates and preserves a fraction of the cytoplasm of a living cell, thereby recording and preserving the transcriptome, proteome, and metabolome of the cell present at the time of assembly of the protein capsule. In some embodiments, the self-assembled protein capsule encapsulates RNA, protein, and metabolites inside the cytoplasm of a cell. In some embodiments, the self-assembled protein capsule protects the encapsulated RNA, protein, and metabolites from degradation in the cell, and during and after isolation of the protein capsule from the cell.

In some embodiments, the self-assembled protein capsule stores RNA, protein, and metabolites. In some embodiments, the self-assembled protein capsule stores RNA, protein, and metabolites encapsulated from the cytoplasm of a cell. In some embodiments, the selfassembled protein capsule stores the encapsulated RNA, protein, and metabolites inside the cytoplasm of the cell. In some embodiments, the self-assembled protein capsule comprises a synthetic nucleocapsid. In some embodiments, the synthetic nucleocapsid has at least two different subunits. In some embodiments, the synthetic nucleocapsid has a net internal positive charge.

In some embodiments, the self-assembled protein capsule can be isolated from a live cell.

Protein Capsule and Subunits

The molecular time capsules (MTCs) used in the methods described herein are selfassembling protein capsules comprised of protein subunits. In some embodiments the selfassembled protein capsule comprises one or more types of protein subunits. In some embodiments, the self-assembled protein capsule comprises a synthetic nucleocapsid that comprises at least two different types of subunits. Examples of subunits and protein capsules include those described in WO 2019/094669 and Butterfield et al. (Nature. 2017 Dec 21;552(7685):415-420).

In some embodiments, the self-assembled protein capsule comprises a natural, evolved, or synthetic viral capsid. Examples of viral capsules include the major capsid protein VP1 of the human JC virus which when expressed in Escherichia coli can selfassemble and which has affinity for both RNA and DNA capture (Ou WC, Wang M, Fung CY, Tsai RT, Chao PC, Hseu TH, Chang D. The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells. J Gen Virol. 1999 Jan;80 (Pt 1 ):39-46. doi: 10.1099/0022-1317-80-1-39. PMID: 9934681.), and Ty3 GAG3, a yeast retrovirus which encodes a capsid, spacer and nucleocapsid and has been shown to be capable of self-assembly in Escherichia coli and which has been shown to interact with RNA (Larsen, L. S., Kuznetsov, Y., McPherson, A., Hatfield, G. W., & Sandmeyer, S. (2008). TY3 GAG3 protein forms ordered particles in Escherichia coli. Virology, 370(2), 223-227. https://doi.Org/10.1016/j.virol.2007.09.017). These viral capsules have also led to the development of synthetic nucleocapsids such as enveloped protein nanocages, which are capable of forming a caged structure with three engineered surfaces. In some embodiments the three engineered surfaces are the interior, the exterior, and the intersubunit.

An important feature of MTCs is compartmentalization and separation between the inside and outside of the MTC such that the RNA (and/or proteins and/or metabolites) inside the MTC is sufficiently separated from the cytoplasm to be protected from decay or exchange with other RNAs (and/or proteins and/or metabolites). This function of the MTCs can be achieved by the existence of separated interior and exterior surfaces, and strong relationships between subunits to prevent exchange and ensure that the capsules remain structured.

In some embodiments, the self-assembled protein capsule comprises natural, evolved, or synthetic protein organelle. Examples of protein organelle include bacterial encapsulins (also referred to as linocin-like proteins) (Wiryaman, T., & Toor, N. (2022). Recent advances in the structural biology of encapsulin bacterial nanocompartments. Journal of structural biology: 6, 100062. https://doi.Org/10.1016/j.yjsbx.2022.100062) and ferritins, selfassembling protein capsules that store and regulate intracellular iron concentrations (Elizabeth C. Theil, Rabindra K. Behera, Takehiko Tosha, Ferritins for chemistry and for life, Coordination Chemistry Reviews, ISSN 0010-8545, https://doi.Org/10.1016/j.ccr.2012.05.013.). The self-assembled protein capsule can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of subunits.

The protein subunits, once expressed in a cell, self-assemble into the protein capsids. Thus, inducing expression of the one or more types of protein subunits at a selected time provides for capturing RNAs, proteins, and/or metabolites present in the cytoplasm of the cell at the selected time.

Expression of protein subunits

The protein subunits that self-assemble into the protein capsule are expressed in live cells from nucleic acid molecules encoding the protein subunits, or vectors (e.g., plasmids, viral genomes) that comprise the nucleic acid molecules. The one or more nucleic acids or vectors encoding the one or more subunits of the self-assembled protein capsule may be delivered to a cell by any methods known in the art for delivering nucleic acids. For example, for delivering nucleic acids to a prokaryotic cell, the methods include, without limitation, transformation, transduction, conjugation, and electroporation. For delivering nucleic acids to a eukaryotic cell, methods include, without limitation, transfection, electroporation, and using viral vectors.

Once present in the cell, the expression of the protein subunits can be induced by inducing transcription and translation of the nucleic acid molecules encoding the protein subunits or by site-specific recombination. The expression can be induced at selected times to capture the transcriptome of the cell present at the selected times. Promoters

Expression of engineered nucleic acids is driven by a promoter operably linked to a nucleic acid containing, for example, a nucleic acid encoding one or more types of protein subunits that form the self-assembling protein capsule. A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. A promoter may also contain subregions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof.

Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.”

In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology.

In some embodiments, the self-assembled protein capsule comprises at least one promoter. In some embodiments, a promoter is an “inducible promoter,” which refer to a promoter that is characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by, or contacted by an inducer signal. An inducer signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical, or non-chemical compound) or protein that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Thus, a “signal that regulates transcription” of a nucleic acid refers to an inducer signal that acts on an inducible promoter. A signal that regulates transcription may activate or inactivate transcription, depending on the regulatory system used. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation of a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

The administration or removal of an inducer signal results in a switch between activation and inactivation of the transcription of the operably linked nucleic acid sequence. Thus, the active state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is actively regulating transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is expressed). Conversely, the inactive state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is not actively regulating transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is not expressed).

In some embodiments, the self-assembled protein capsule comprises at least one inducible promoter. In some embodiments, one or more inducible promoter is operably linked to one or more sequence encoding the one or more type of protein subunit. In some embodiments, the inducible promoter is a chemically-inducible promoter, a temperatureinducible promoter, or a light-regulated promoter. In some embodiments the inducible promoter is a tissue specific promoter.

An inducible promoter of the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in light, pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s). An extrinsic inducer signal or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones, or combinations thereof.

Inducible promoters of the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, isopropyl B-D-l -thiogalactopyranoside (IPTG) promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

In some embodiments, an inducer signal of the present disclosure is isopropyl -D-1- thiogalactopyranoside (IPTG), which is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator. IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of 0-galactosides into monosaccharides. The sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer. IPTG is an effective inducer of protein expression, for example, in the concentration range of 100 pM to 1.0 mM. Concentration used depends on the strength of induction required, as well as the genotype of cells or plasmid used.

In some embodiments, inducible promoters of the present disclosure are from prokaryotic cells (e.g., bacterial cells). Examples of inducible promoters for use prokaryotic cells include, without limitation, bacteriophage promoters (e.g., Plslcon, T3, T7, SP6, PL) and bacterial promoters (e.g., Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, Pm), or hybrids thereof (e.g., PLlacO, PLtetO). Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters such as positively regulated c70 promoters (e.g., inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promoter, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), cS promoters (e.g., Pdps), c32 promoters (e.g., heat shock) and c54 promoters (e.g., glnAp2); negatively regulated E. coli promoters such as negatively regulated c70 promoters (e.g., Promoter (PRM+), modified lamdba Prm promoter, TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLacOl, dapAp, FecA, Pspac-hy, pci, plux-cl, plux-lac, CinR, CinL, glucose controlled, modified Pr, modified Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR_regulated, Betl_regulated, pLac_lux, pTet_Lac, pLac/Mnt, pTet/Mnt, LsrA/cI, pLux/d, LacI, LacIQ, pLacIQl, pLas/d, pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa_R0011, LacI/ara- l , pLadq, rmB Pl, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF, RcnR), GS promoters (e.g., Lutz-Bujard LacO with alternative sigma factor G38), G32 promoters (e.g., Lutz-Bujard LacO with alternative sigma factor G32), and G54 promoters (e.g., glnAp2); negatively regulated B. subtilis promoters such as repressible B. subtilis GA promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and GB promoters. Other inducible microbial promoters may be used in accordance with the present disclosure.

In some embodiments, the timing and duration of the RNA, protein, and/or metabolite encapsulation by the self-assembled protein capsule is controlled by adding to the live cell and subsequently withdrawing from the live cell the inducer of the inducible promoter.

Purification Tags

The protein subunits can be modified in various ways. In some embodiments, the protein subunits are modified to include a purification tag. In some embodiments the purification tag is a histidine tag.

A histidine tag is an amino acid motif that consists of two or more histidine residues at either the N- or C- terminus of a protein. In some embodiments, the self-assembled protein capsule comprises at least one histidine tag. In some embodiments, the self-assembled protein capsule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more histidine tags. The histidine tags can be attached to one or more types of the protein subunits of the protein capsule. In some embodiments, the histidine tag comprises at least two histidine residues. In some embodiments, the histidine tag comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In some embodiments, the histidine tag comprises more than 10 residues. In some embodiments, the histidine tag comprises 6 histidine residues.

In some embodiments, the purification tag is a human influenza hemagglutinin (HA) tag. In some embodiments, the purification tag is a FLAG tag. In some embodiments, the purification tag is a polyarginine tag (poly-ARG or nARG tag). In some embodiments, the purification tag is a glutathione-S-transferase (GST) tag. In some embodiments, the purification tag is a streptavidin-binding peptide (SBP). In some embodiments, the purification tag is a streptavidin-binding tag (Strep-tag). In some embodiments, the purification tag is a strep-tag II (modified streptavidin-binding tag). In some embodiments, the purification tag is a calmodulin binding peptide. In some embodiments, the purification tag is a chitin-binding tag. In some embodiments, the purification tag is a maltose-binding tag. In some embodiments, the purification tag is a cellulose-binding tag.

In some embodiments, the histidine tag or other purification tag is at the N-terminus of one or more types of protein subunits. In some embodiments, the histidine tag or other purification tag is at the C-terminus of one or more types of protein subunits.

Proteins that have a histidine tag or other purification tag can be isolated by affinity chromatography. In some embodiments, the self-assembled protein capsule is separated from a cell by affinity chromatography. For example, the cell can by lysed by addition of chemicals or application of physical forces to produce a cell lysate. The cell lysate can then be contacted with an appropriate affinity chromatography material (e.g., a resin) that binds the purification tag to isolate the protein capsules from the cell lysate. The affinity chromatography material can be added to the cell lysate, or the cell lysate can be flowed over the affinity chromatography material, such as in a column. Following contact with the affinity chromatography material, the protein capsules can be isolated by removing the affinity chromatography material from the lysate or by altering the conditions of the mixture containing the affinity chromatography material to reduce or eliminate binding between the protein capsules and the affinity chromatography material.

In some embodiments, the self-assembled protein capsule is separated from a cell by metal affinity chromatography. In metal affinity chromatography proteins are separated according to their affinity for metal ions which are immobilized on a resin matrix. In some embodiments, the metal ions immobilized in a resin matrix in metal affinity chromatography are Zn²⁺, Cu²⁺, Cd²⁺, Hg²⁺, Co²⁺, Ni²⁺, or Fe²⁺. In some embodiments, the metal ions immobilized in a resin matrix in metal affinity chromatography are Ni²⁺. In some embodiments, the metal ions immobilized in a resin matrix in metal affinity chromatography are Co²⁺.

In some embodiments, the metal affinity chromatography uses a nickel chromatography resin. In some embodiments, the metal affinity chromatography uses a cobalt chromatography resin.

RNA Isolation and Analysis

In some embodiments, the RNA encapsulated by the self-assembled protein capsule is isolated from the self-assembled protein capsule. In some embodiments, the RNA is isolated from the self-assembled protein capsule by any suitable method known in the art. Methods of RNA isolation include, but are not limited to; organic extraction methods, spin basket formats, magnetic particle methods, and direct lysis methods.

In some embodiments, the isolated RNA is detectable. In some embodiments the isolated RNA is quantified by any suitable method known in the art. Methods of RNA quantification include, but are not limited to; sequencing, UV spectroscopy, RT-PCR, and fluorometric methods.

In some embodiments, the isolated RNA is sequenced. In some embodiments, the isolated RNA is sequenced by any suitable method known in the art. In some embodiments, the isolated RNA is sequence by RNA-seq.

Protein Isolation and Analysis

In some embodiments, the protein is isolated from the self-assembled protein capsule by any suitable method known in the art. Methods of protein isolation include, but are not limited to; size exclusion (e.g., gel filtration chromatography), ion-exchange chromatography, free-flow electrophoresis, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and non-denaturing-condition electrophoresis.

In some embodiments, the isolated protein is detectable. In some embodiments the isolated protein is quantified by any suitable method known in the art. Methods of protein quantification include, but are not limited to: UV absorption, enzyme linked immunosorbent assay (ELISA), bicinchoninic acid assay (BCA), high-performance liquid-based chromatography (HPLC), the use of fluorescently labelled or radio-chemically labelled proteins, western blot, and mass spectrometry.

In some embodiments, the isolated protein is sequenced. In some embodiments, the isolated protein is sequenced by any suitable method known in the art. In some embodiments, the isolated protein is sequenced by mass spectrometry or Edman degradation.

Metabolite Isolation and Analysis

In some embodiments, the metabolite encapsulated by the self-assembled protein capsule is isolated from the self-assembled protein capsule. In some embodiments, the metabolite is isolated from the self-assembled protein capsule by any suitable method known in the art. Methods of metabolite isolation include but are not limited to; gas chromatography (GC), high-performance liquid chromatography (HPLC), ultraperformance liquid chromatography (UPLC), capillary electrophoresis (CE), and western blot. In some embodiments, the isolated metabolite is detectable. In some embodiments the isolated metabolite is quantified by any suitable method known in the art. Methods of metabolite quantification include but are not limited to; mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and ELISA.

Cells

Also provided herein are cells comprising the nucleic acid or the vectors encoding the self-assemble protein capsule as described herein. A “cell” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular.

In some embodiments, a cell for use in accordance with the present disclosure is a prokaryotic cell, which may comprise a cell envelope and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. In some embodiments, the cell is a bacterial cell. As used herein, the term “bacteria” encompasses all variants of bacteria, for example, prokaryotic organisms and cyanobacteria. Bacteria are small (typical linear dimensions of around 1 micron), non-compartmentalized, with circular DNA and ribosomes of 70S. The term bacteria also include bacterial subdivisions of Eubacteria and Archaebacteria. Eubacteria can be further subdivided into gram-positive and gram- negative Eubacteria, which depend upon a difference in cell wall structure. Also included herein are those classified based on gross morphology alone (e.g., cocci, bacilli). In some embodiments, the bacterial cells are gram-negative cells, and in some embodiments, the bacterial cells are gram-positive cells. Examples of bacterial cells that may be used in the methods described herein include, without limitation, cells from Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp ., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp. In some embodiments, the bacterial cells are Escherichia coli cells.

In some embodiments, a cell for use in accordance with the present disclosure is a eukaryotic cell, which comprises membrane-bound compartments in which specific metabolic activities take place, such as a nucleus. Examples of eukaryotic cells for use in the methods described herein include, without limitation, mammalian cells, insect cells, yeast cells (e.g., Saccharomyces cerevisiae) and plant cells. In some embodiments, the eukaryotic cells are from a vertebrate animal. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is from a rodent, such as a mouse or a rat. Examples of vertebrate cells for use in accordance with the present disclosure include, without limitation, reproductive cells including sperm, ova and embryonic cells, and non-reproductive cells including, immune, kidney, lung, spleen, lymphoid, cardiac, gastric, intestinal, pancreatic, muscle, bone, neural, brain and epithelial cells.

In some embodiments, a cell used in accordance with this disclosure is a stem cell, including embryonic stem cells or induced pluripotent stem cells.

In some embodiments, the cell is a live cell. In some embodiments, the cell is a diseased cell. A “diseased cell” as used herein refers to a cell whose biological functionality is abnormal, compared to a non-diseased (normal) cell.

EXAMPLES

Example 1: Methods

Cell Strains:

Escherichia coli (E. coli) strain K-12 MG1655 and Bacillus subtilis (B. sublilis) strain 168.

T ransformation :

For transformation into Zymo Mix & Go DH5 Alpha competent cells (Zymo T3007) lOOuL of cells were thawed on ice per reaction. Then l-4pL plasmid DNA was added followed by gentle mixing. After a 5-minute incubation on ice cells 400pL of prewarmed SOB was added and the mixture was incubated in an Eppendorf tube for 1 hour at 37C with 300 RPM shaking. The mixture was then spread on pre-warmed agar plates with the appropriate antibiotic.

For transformation into non-competent wild-type MG 1655 E. coli the cells were made competent using the TSS protocol (11). 3mL of LB was inoculated with a bacterial colony from a fresh agar plate, followed by an incubate at 37C and spun at 230 RPM for 1.5 to 2 hours. 200pL of cells were then added to 200pL ice cold TSS buffer (LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5) and IpL plasmid. The cell solution was then vortexed and incubated on ice for 20-30 minutes, followed by an incubation for 45-60 min at 37C on the thermomixer, while shaking at 900 RPM. The cells were then plated on the appropriate antibiotic. Plasmids: pMP026 (FIG. 8) is a Kan resistant plasmid which contains constitutively produced lacl and IPTG inducible transcript encoding the pentameric and trimeric units of the selfassembling protein capsule that serves as the Molecular Time Capsule. These units are derived from the I53-50-v4N genome from Butterfield et at 2017 (https://doi.org/10.1038/nature25157). The full plasmid map can be found on https://github.com/gwlilabmit/MTC_2023_Scripts. This plasmid was constructed using the method described below. It was inserted into the E. coli strain K-12 MG1655 using the method outlined in greater detail below. This pMP026-containing E. coli strain was the one used for all experiments in this work.

Plasmid Assembly:

Plasmid components were either PCRed from a pre-existing plasmid, followed by Dpnl (NEB #R0176L) digestion or were ordered directly as gene blocks from IDT. Plasmids were constructed by Gibson assembly, using the protocol and reagents associated with NEB #E2611 with a total volume of 4pL. Plasmids were then transformed into Zymo Mix & Go DH5 Alpha Competent Cells (Zymo T3007) for verification and amplification before repurification and transformation into wild type MG1655 E. coli. All plasmid purification was done using overnight culture and the Zymo Zyppy Plasmid Miniprep Kit (Genesee 11-30). Plasmids were verified using Sanger sequencing, or by Plasmidsaurus.

Media:

All experiments were conducted using Luria Broth (LB).

Protein Purification:

Harvested Cell Pellets were re-hydrated in 40mL Lysis Buffer (150mM Imidazole, 250 mM NaCl, 25mM Tris-HCL, pH 8, with Protease Inhibitors) and the OD 600 values of each rehydrated solution were measured. Each sample was then sonicated using the 450W Ultrasonic Homogenizer (10 to 300mL) from US Solid for a program of 2 seconds on at 25% power, followed by a 6 second off break for a total of 10 minutes. Lysate was then clarified by centrifugation at 4C using a Sorvall RC-5B Refrigerated Superspeed Centrifuge at 10,000 RPM for 45 min, after which the clarified supernatant was retained for loading on the protein column. To prepare the protein column 2 mL (ImL column volume) of Nickel NTA resin was loaded onto the 5mL Polypropylene columns from Qiagen and the resin buffer was allowed to drain. The resin was then rinsed with 15 column volumes of ddH2O followed by 15 column volumes of wash buffer (150mM Imidazole, 250 mM NaCl, 25mM Tris-HCL, pH 8) to equilibrate the column. After this the clarified supernatant is loaded onto the column. The column is then washed with 15 column volumes of the wash buffer followed by an elution with the elution buffer (500mM Imidazole, 250 mM NaCl, 25mM Tris-HCL, pH 8). 3 column volumes of the elution buffer were used, but only the last 2 column volumes were kept. The purified proteins are then treated with RNase A (IpL/mL 20C for 10 minutes) before proceeding with RNA extraction.

RNA Extraction:

Add a prewarmed (65C) solution of 500mL phenol acid chloroform with 29pL 20% SDS to 500mL of sample to a 1.5mL Eppendorf in a thermomixer (split between multiple tubes if needed). Incubate at 65C for 5 minutes at 1,400 RPM, followed by a 5-minute incubation of the samples on ice. Spin the samples at 20,000g for 2 minutes then transfer the top aqueous layer to a new non-stick tube. Add 45pL 3M NaAc (pH 5.4), 500mL 100% Isopronal and IpL GlycoBlue coprecipitant. Chill the tubes at -80C for 30 min+ (possible stopping point) and then spin at 20,000g for 60 min at 4C. Remove and discard the supernatant then add 250 mL pre-chilled 80% Ethanol and spin at 20,000 g for 5 min. Remove and discard the supernatant then resuspend the samples in lOOpL 10 mM Tris 7. Process the sample using the modified version of the Zymo-5 RNA Clean and Concentrator columns to remove all RNAs < 200 nt (to remove tRNAs). Then elute the sample in 85 pL DEPC-water and proceed immediately with DNase treatment.

DNase Treatment:

Add lOpL lOx Turbo DNAse buffer and 5pL Turbo DNase to 85pL RNA. Incubate for 20-30 min at 37 C in Eppendorf thermomixer without shaking. Add 330 pL 100% Ethanol, l lpL 3M NaAc (pH 5.4) and 1 pL GlycoBlue coprecipitant. Chill the tubes at -80C for 30 min+ (possible stopping point) and then spin at 20,000G for 60 min at 4C. Remove and discard the supernatant then add 250 mL pre-chilled 80% Ethanol and spin at 20,000 g for 5 min. Remove and discard the supernatant then resuspend the samples in 13.5pL DEPC- water. Measure concentrations using High Sensitivity RNA kit for Qubit, then proceed with library preparation. Library Preparation:

Libraries in this paper were prepared using NEB’s rRNA Depletion Kit (Bacteria) for rRNA removal with beads (NEB #E7860). Post rRNA removal samples were prepared for either Illumina or Singular Sequencing using NEBNext Ultra II RNA Library Prep Kit for Illumina with Beads (NEB #E7775). Singular specific primers were used for the libraries sequencing on Singular which had the S1/S2 handles instead of Illumina’s p7/p5 handles.

Library Analysis:

All fastq files were trimmed using seqtk and cutadapt to remove bases of low quality and adapters. Reads were then aligned using bowtie27, after which the density of the 5’ ends was quantified using samtools28 and the CDS files for each genome were used to quantify how many transcripts were found within each gene. The complete scripts for raw analysis, as well as processed data files (in the format of counts per gene) can be found on https ://github .com/gwlilabmit/MTC_2023_Scripts .

Example 2: Comparison between MTC-encapsulated RNA and total cellular RNA

To assess the ability of MTCs to capture and store RNA, cell lysates and MTC content were collected from the same E. coli culture. MG1655 E. coli cells containing histidine tagged MTCs and a Kan marker, were streaked on a Kan marker plate, and left to grow overnight at 37C. The next day 3 single colonies were selected from the plates and incubated in separate test tubes with 5mL Luria Broth (LB) and Kan for 2 hours. The cultures were then back diluted to allow for 12 doublings before reaching an optical density (OD) of 0.3 in 500mL of pre-warmed LB with ImM IPTG and Kan. Before reaching the OD of 0.3 both the lysate and MTC samples were harvested. The MTC samples were harvested by splitting the volume between four 50mL Falcon Tubes and spinning them down for 10 min at 4000 RPM at 4C in an Eppendorf 5810R Centrifuge. The supernatant was discarded, and the cell pellets were frozen for future protein purification. The lysate sample was collected by placing 500pL of culture directly into prewarmed RNA extraction solution and proceeding with RNA extraction.

The histidine tagged MTCs were purified using Ni-NTA columns from exponential phase cells that have been expressing capsule proteins steadily (for >10 generations). Cells were resuspended in a buffer containing 150mM Imidazole before being added to the column and washed with a 150mM Imidazole buffered solution (FIG. 3). The full description of the purification method can be found in Example 1. Expression of MTCs only slightly increases the population doubling time (from 20 minutes to 28.5 minutes) (FIG. 4). 2.8*10"⁴+/-6.7*10'⁵ fg of RNA per cell was recovered from cells expressing MTCs, compared to wild type E. coli cells, which had an undetectable amount of RNA recovered when using the same method. When comparing the RNA captured by the MTC and the lysate sample, they showed comparable levels of mRNA on a gene-by-gene basis across replicates (R=0.993 between replicates 1 and 2, R=0.970 between replicates 1 and 3, and R=0.983 between replicates 2 and 3) (FIG. 1C and FIGs. 5A-5F). The mRNA levels in the total cell lysate also correlated with the MTC samples, albeit less well compared to the reproducibility of the MTC capture across biological replicants (median R= versus R=) (FIGs. 5A-5F and FIG. 6). Among genes with >100 reads, the standard deviation for their RNA ratios between the lysate and MTC samples is 1.34-fold (FIG. 1C). In addition, the capture bias is highly reproducible across replicates (FIG. IB), which enables differential expression analysis using MTCs collected from different samples. Interestingly, the total mRNA is slightly enriched over ribosomal RNA inside MTCs, increasing from 10% of reads in the lysate sample to 33% in MTC, likely reflecting the fact that intact ribosomes are excluded from the MTCs due to their physical dimensions. The majority of the RNA fragments recovered from MTCs are shore (<200nt), suggesting that MTC-based capture leverages the abundant RNA decay intermediates recently reported to dominate the transcriptome (24) (FIGs. 7A-7B).

Next, to determine if MTCs could be cleanly extracted from a subpopulation of cells without substantial contamination from RNA in the bulk lysate, MG1655 E. coli cells containing histidine tagged MTCs and a Kan marker cells were cultured along with B. subtilis cells, which did not contain MTCs (FIG. 2A). The cells were cultured in a ratio of 1:1. The species of bacteria used, E. coli and B. subtilis, have distinct genomes, facilitating the identification of which species a particular RNA originates, after RNA-seq analysis.

MP050 cells were streaked from a glycerol stock onto a LB and Kan marker plate and left to grow overnight. A colony from this plate was selected the next day and added to lOmL LB with Kan and grown overnight at 37C on a shaker plate, shaking at approximately 225 RPM. This overnight culture was then diluted to achieve 12 doublings before reaching OD 0.3 in pre-warmed LB with Kan. The culture was then grown at 37 C on a shaker plate, shaking at approximately 225 RPM. At OD of 0.3 ImM IPTG was added. At OD of 2 the culture was divided between 50mL Falcon tubes and spun for 10 minutes at 4000 RPM in a pre-chilled (4C) Eppendorf 5810R Centrifuge. The Supernatant was then discarded, and the cell pellets were flash frozen in liquid nitrogen and stored at -80C. The B. subtilis cells were inoculated directly from a glycerol stock into 5mL LB, then grown for 2 hours at 37C on a rotator spinning ~ 225 RPM. This culture was then diluted to achieve 12 doublings before reaching OD of 0.3 in pre-warmed LB. At OD of 2 the culture was divided between 50mL Falcon tubes and spun for 10 minutes at 4000 RPM in an Eppendorf 5810R Centrifuge. The supernatant was then discarded, and the cell pellets were flash frozen in liquid nitrogen and stored at -80C.

After co-culture the cells were harvested and evaluated using RNA-seq. The heterogenous lysate contained 47% RNA that was mapped to B. subtilis, while the MTCs extracted from the same lysate contained 99.97% RNA from E. coli (FIG. 2B and 2C). The level of B. subtilis RNA mapped in the MTCs (0.03%) is close to that of the baseline level estimated using an E. coli RNA sample sequenced simultaneously (0.1%). This indicates that MTC-purification procedure specifically captures the encapsulated RNAs, and that the transcriptome of a targeted subpopulation can be successfully isolated using the MTC without contamination from non-encapsulated transcripts.

Example 3: Assessing the stability of encapsulated RNAs over time

To determine if MTCs provide prolonged transcriptome storage inside host cells, the maintenance of MTC-uncalculated RNA after shifting cells to a different environment that would alter the host cell’s transcriptomics was examined. First, MG1655 E. coli cells containing histidine tagged MTCs and a Kan marker were streaked on a Kan marker plate and grown overnight at 37C. The next day single colonies were selected from the plates and incubated in a test tube with 5mL LB and Kan for 2 hours. The culture was then back diluted in a 500mL volume with Kan to achieve 10 doublings before reaching an OD of 0.03. Once the OD was reached, IPTG was added, for a final concentration of ImM. 1 hour later the capsule induction was shut off by filtering the cells. The filter was washed with half the original cell volume of warm LB. The cells were then resuspended in the original volume of LB without any antibiotics or IPTG. 15 minutes post wash the pre-stress lysates and MTC were collected. The MTC sample was collected by filtering 2 volumes of 125mL of culture. The cells were then resuspended in two 50mL falcon tubes in pre chilled (4C) LB. The resuspension was then centrifuged for 10 min at 4000 RPM at 4C in an Eppendorf 5810R centrifuge. The supernatant was discarded, and the cell pellets were frozen for future protein purification. The lysate sample was collected by placing 500pL of culture directly into prewarmed RNA. extraction solution and then proceeded to RNA extraction (FIG. 2D). 4% ethanol was then added as a stressor to the remaining 250mL of culture. The remaining culture was then allowed to grow for 45 minutes before having the post-stress lysate and MTC samples harvested (FIG. 2D). Post-stress lysate and MTC samples were harvested in a manner similar to the one previously described.

After the stress, the host transcriptome underwent substantial remodeling, with 81 genes changing by >10 fold and an overall Pearson correlation coefficient of R=0.79 (FIG. 2E), as seen in the pre- and post-stress lysate samples. By contrast, the MTC samples collected pre- and post-stress have similar RNA levels, with no genes changing by >10 fold, and an overall Pearson correlation coefficient of R=0.99 (FIG. 2E). The most extremely shifted gene in the lysate sample, tnaA, changed by 124-fold, whereas this same gene only changed by 1.5-fold in the MTCs. These results demonstrate that MTC contents remain static despite large contextual changes in the cell state. These results show that MTCs can capture and preserve high-fidelity snapshots of the transcriptome in living cells.

References

1. Shaffer, S. M. et al. Memory Sequencing Reveals Heritable Single-Cell Gene Expression Programs Associated with Distinct Cellular Behaviors. Cell 182, 947- 959.el7 (2020).

2. Xia, C., Fan, J., Emanuel, G., Hao, J. & Zhuang, X. Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression. Proc. Natl. Acad. Sci. U. S. A. 116, 19490-19499 (2019).

3. Zhang, J., Han, X., Ma, L., Xu, S. & Lin, Y. Deciphering a global source of non- genetic heterogeneity in cancer cells. Nucleic Acids Res. 51, 9019-9038 (2023).

4. Sampaio, N. M. V., Blassick, C. M., Andreani, V., Lugagne, J.-B. & Dunlop, M. J.

Dynamic gene expression and growth underlie cell-to-cell heterogeneity in Escherichia coli stress response. Proc. Natl. Acad. Sci. U. S. A. 119, e2115032119 (2022).

5. Hwang, B., Lee, J. H. & Bang, D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp. Mol. Med. 50, 1-14 (2018).

6. Saliba, A.-E., Westermann, A. J., Gorski, S. A. & Vogel, J. Single-cell RNA-seq: advances and future challenges. Nucleic Acids Res. 42, 8845-8860 (2014).

7. Shalek, A. K. et al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510, 363-369 (2014).

8. Jovic, D. et al. Single-cell RNA sequencing technologies and applications: A brief overview. Clin. Transl. Med. 12, e694 (2022).

9. La Manno, G. et al. RNA velocity of single cells. Nature 560, 494-498 (2018). 10. Bergen, V., Lange, M., Peidli, S., Wolf, F. A. & Theis, F. J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat. Biotechnol. 38, 1408-1414 (2020).

11. Qiu, X. et al. Mapping transcriptomic vector fields of single cells. Cell 185, 690- 711.e45 (2022).

12. Bergen, V., Soldatov, R. A., Kharchenko, P. V. & Theis, F. J. RNA velocity-current challenges and future perspectives. Mol. Syst. Biol. 17, el0282 (2021).

13. Lin, D. et al. Time-tagged ticker tapes for intracellular recordings. Nat. Biotechnol. 41, 631-639 (2023).

14. Linghu, C. et al. Recording of cellular physiological histories along optically readable self-assembling protein chains. Nat. Biotechnol. 41, 640-651 (2023).

15. Chen, W. et al. Live-seq enables temporal transcriptomic recording of single cells. Nature 608, 733-740 (2022).

16. Homs, F. et al. Engineering RNA export for measurement and manipulation of living cells. Cell (2023) doi:10.1016/j.cell.2023.06.013.

17. Schmidt, F., Cherepkova, M. Y. & Platt, R. J. Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 562, 380-385 (2018).

18. Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature 608, 217-225 (2022).

19. Jiao, C. et al. RNA recording in single bacterial cells using reprogrammed tracrRNAs. Nat. Biotechnol. (2023) doi:10.1038/s41587-022-01604-8.

20. Silas, S. et al. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas 1 fusion protein. Science 351, aad4234 (2016).

21. Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aafl l75 (2016).

22. Schmidt, F. et al. Noninvasive assessment of gut function using transcriptional recording sentinel cells. Science 376, eabm6038 (2022).

23. Shalon, D. et al. Profiling the human intestinal environment under physiological conditions. Nature 617, 581-591 (2023).

24. Butterfield, G. L. et al. Evolution of a designed protein assembly encapsulating its own RNA genome. Nature 552, 415-420 (2017).

25. Herzel, L., Stanley, J. A., Yao, C.-C. & Li, G.-W. Ubiquitous mRNA decay fragments in E. coli redefine the functional transcriptome. Nucleic Acids Res. 50, 5029-5046 (2022). 26. Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences 86, 2172-2175 (1989).

27. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

28. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).

29. Ou, W. C. et al. The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells. J. Gen. Virol. 80 ( Pt 1), 39-46 (1999).

30. Votteler, J. et al. Designed proteins induce the formation of nanocage-containing extracellular vesicles. Nature 540, 292-295 (2016).

31. Larsen, L. S. Z., Kuznetsov, Y., McPherson, A., Hatfield, G. W. & Sandmeyer, S.

TY3 GAG3 protein forms ordered particles in Escherichia coli. Virology 370, 223- 227 (2008).

32. Theil, E. C., Behera, R. K. & Tosha, T. Ferritins for Chemistry and for Life. Coord. Chem. Rev. 257, 579-586 (2013).

33. Wiryaman, T. & Toor, N. Recent advances in the structural biology of encapsulin bacterial nanocompartments. J Struct Biol X 6, 100062 (2022).

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A method for monitoring RNA gene, protein, and/or metabolite expression in a live cell comprising expressing in the live cell one or more type of protein subunit that selfassemble into a protein capsule, wherein contents of the cytoplasm of the live cell are encapsulated by the self-assembled protein capsule and isolating the self-assembled protein capsule from the live cell.

2. The method of claim 1, wherein the self-assembled protein capsule is a synthetic nucleocapsid, evolved nucleocapsid, or synthetic viral capsid.

3. The method of claim 1 or claim 2, wherein the self-assembled protein capsule has a net internal positive charge.

4. The method of claim 1 or claim 2, wherein the self-assembled protein capsule has a net internal negative charge.

5. The method of any one of claims 1 to 4, wherein the self-assembled protein capsule comprising at least two different types of subunits.

6. The method of claim 2, wherein the synthetic viral capsid is the major capsid protein VP1 of the human JC virus or Ty3 GAG3.

7. The method of any one of claims 1 to 6, wherein the self-assembled protein capsule comprises one or more natural, evolved, or synthetic protein organelles.

8. The method of claim 7, wherein the protein organelle is a bacterial encapsulin or a ferritin.

9. The method of any one of claims 1-8, wherein the self-assembled protein capsule captures RNA, protein, and/or metabolite inside the cytoplasm of the cell.

10. The method of claim 9, wherein the wherein the self-assembled protein capsule captures RNA inside the cytoplasm of the cell.

11. The method of any one of claims 1-10, wherein the self-assembled protein capsule stores the RNA, protein, and/or metabolite inside the cytoplasm.

12. The method of any one of claims 1-11, wherein the self-assembled protein capsule stores RNA inside the cytoplasm.

13. The method of any one of claims 1-12, further comprising isolating the RNA, protein, and/or metabolite from the isolated self-assembled protein capsule.

14. The method of any one of claims 1-13, further comprising isolating the RNA from the isolated self-assembled protein capsule.

15. The method of claim 14, further comprising sequencing the isolated RNA.

16. The method of any one of claims 1-15, wherein one or more inducible promoter is operably linked to one or more sequence encoding the one or more type of protein subunit.

17. The method of claim 16, wherein the inducible promoter is a chemically-inducible promoter, a temperature-inducible promoter, or a light-regulated promoter.

18. The method of claim 17, wherein the chemically-inducible promoter is an isopropyl P- D-l -thiogalactopyranoside promoter (IPTG), an alcohol-regulated promoter, a tetracycline- regulated promoter, a steroid-regulated promoter, a metal-regulated promoter.

19. The method of claim 16, wherein the inducible promoter is a tissue specific promoter, a pathogenesis-regulated promoter, a bacteriophage promoter, a bacterial promoter, or a hybrid of a bacteriophage and bacterial promoter.

20. The method of any one of claims 16-19, wherein the timing and duration of the RNA, protein, and/or metabolite encapsulation by the self-assembled protein capsule is controlled by adding to the live cell and subsequently withdrawing from the live cell the inducer of the inducible promoter.

21. The method of any one of claims 1-20, wherein the one or more protein subunits of the self-assembled protein capsule comprises at least one purification tag.

22. The method of claim 21, wherein the purification tag is a histidine tag, polyarginine tag, GTS tag, FLAG tag, SBP tag, strep-tag II, calmodulin binding protein, chitin-binding tag, maltose-binding tag, or cellulose-binding tag.

23. The method of claim 22, wherein the purification tag is a histidine tag.

24. The method of claim 23, wherein the histidine tag is at the N-terminus of the one or more types of protein subunits.

25. The method of claim 23 or claim 24, wherein the histidine tag is at the C-terminus of the one or more types of protein subunits.

26. The method of any one of claims 21 to 25, wherein the self-assembled protein capsule encapsulating RNA, protein, and/or metabolite from the live cell is separated from the live cell by metal affinity chromatography, optionally by binding to a nickel chromatography resin.

27. The method of any one of claims 1-26, wherein the cell is a prokaryotic cell or eukaryotic cell.

28. The method of claim 27, wherein the cell is a bacterial cell.

29. The method of claim 28, wherein the cell is an E. coli cell.

30. The method of claim 27, wherein the cell is a mammalian cell.

31. The method of claim 30, wherein the cell is a human cell.