Patent Application
20250075241
The present disclosure relates to the protection of linear deoxyribonucleic acid (DNA) molecules from exonucleolytic degradation in a biology environment and to methods of producing proteins from linear expression templates.
Over the past two decades cell-free synthetic biology has established itself as a versatile platform for advancing biological research at a pace unattainable by traditional cell-based methods. From in vitro expression of proteins for structural [1,2] and microarray analyses, [3] to rapid prototyping of enzymatic [4,5] and regulatory elements, [6-8] to field-deployable diagnostics, [9] and even industrial-scale biomanufacturing of therapeutics, cell-free systems (CFSs) have revolutionised the discovery process across many research disciplines. These CFSs typically consist of extracted cellular transcription-translation (TXTL) machinery in a mixture with energy regeneration and biosynthesis micro components. The great majority of these applications are reliant on Escherichia coli (E. coli) as their chassis organism for extract preparation. [11] Although, particularly in recent years, Vibrio natriegens (V. nat) has also been championed by multiple research laboratories as the next-generation chassis organism due to its faster growth cycle and desirable biosafety level. [12-16]
These bacterial CFSs are known to rely strictly on circular DNA input, as linear expression templates (LETs) with free termini are highly susceptible to exonucleolytic degradation by endogenous enzymes present in the extracts. [17] In E. coli, the helicase exonuclease complex RecBCD has been well-reported to degrade linear DNA with high processivity. [18] Endogenous exonucleases in V. nat extracts are similarly capable of degrading LETs. [16,19] Therefore, despite being extremely useful and advantageous compared to in vivo techniques, this incompatibility with linear DNA means that the huge potential of CFSs is yet to be fully tapped. For instance, cloning and preparation of plasmid templates for a gene of interest can take a minimum of two days by standard laboratory practices; whereas the same gene could be prepared as a LET by Polymerase Chain Reaction (PCR) within three hours and at a far higher throughput. Therefore, by simply replacing plasmids with LETs in cell-free applications a considerable amount of time, cost and labour could be spared to accelerate and expand the discovery process in both fundamental and applied research.[20]
Recognising this potential, various research laboratories have so far tried to develop methods to protect LETs in bacterial extracts.[2,17,19,21-25] In E. coli CFSs, one of the most effective and widely used solutions is the Lambda GamS protein which specifically inhibits RecBCD thereby prolonging the lifetime of LETs in the extract. However, despite some recent efforts,[16, 19,25] the same techniques that work for E. coli are either ineffective or poorly functional in V. nat extracts-likely due to its divergent DNA degradation mechanisms.[14]
The “Tus-Ter” E. coli DNA replication termination system, which has homologues across many γ-proteobacterial strains [28], involves a protein module—the “Tus” protein, and a 23 base pair cognate DNA sequence module—the “Ter” sequence, with a remarkable equilibrium binding constant (KD) of 3.4×10−13 M.28 The high-affinity binding of Tus to the Ter sequence strongly inhibits the progress of helicase-containing complexes towards any DNA sequence preceding the Ter site [29,30] even in eukaryotic systems [31]. As such, the Tus-Ter system has been proposed as a system to regulate replication fork arrest and can be utilized for disrupting DNA replication.
In one embodiment, the present disclosure is a linear double stranded deoxyribonucleic acid (dsDNA) molecule comprising operatively linked in the 5′ to 3′ direction: a) one or more Ter sites at the 5′ terminus (“5′ Ter”); b) a segment comprising DNA sequence of interest; and c) one or more Ter sites at the 3′ terminus (“3′ Ter).
In one embodiment of the linear dsDNA molecule, the DNA sequence of interest is a functional DNA sequence.
In another embodiment of the linear dsDNA molecule, the functional DNA sequence is a gene, a regulatory sequence, a splice site a binding site, a primer, an aptamer or combinations thereof.
In another embodiment of the linear dsDNA molecule, the 3′ Ter is downstream a terminator sequence.
In another embodiment of the linear dsDNA molecule, the DNA sequence of interest is a coding sequence for encoding an expression product and the terminator sequence is located after a STOP codon of the DNA coding sequence and before the 3′ Ter.
In another embodiment of the linear dsDNA molecule, the linear dsDNA molecule further comprises a 5′ DNA buffer region upstream the 5′ end of the DNA sequence of interest and a 3′ DNA buffer region 3′ end downstream the DNA sequence of interest, and wherein the 5′ DNA buffer region includes between 0 to 300 base pairs and the 3′ DNA buffer region includes between 0 to 125 base pairs.
In another embodiment of the linear dsDNA molecule, the DNA sequence of interest is the coding sequence as defined in claim 5, and wherein the 3′ DNA buffer ranges between 45 and 125 base pairs.
In another embodiment of the linear dsDNA molecule, the linear dsDNA further comprises a Tus protein bound to the 5′ Ter site and another Tus protein bound to the 3′ Ter.
In another embodiment of the linear dsDNA molecule, at least one of the one or more Ter sites comprises SEQ ID NO:1.
In another embodiment of the linear dsDNA molecule, the one or more Ter sites at the 5′ terminus comprises SEQ ID NO: 2 and the one or more Ter sites at the 3′ terminus comprises SEQ ID NO: 3.
In another embodiment, the present disclosure relates to a method of protecting a linear deoxyribonucleic acid (DNA) molecule having a free 5′ terminus and a free 3′ terminus from exonuclease degradation comprising: a) adding one or more Ter sites at the free 5′ terminus (“5′ Ter) of the DNA molecule and adding one or more Ter sites at the 3′ terminus (“3′ Ter”) of the DNA molecule, and b) binding a Tus protein to each of the 5′ Ter and the 3′ Ter.
In one embodiment of the method of protecting the linear DNA molecule, the linear DNA molecule is a double stranded deoxyribonucleic acid (DNA) molecule.
In another embodiment of the method of protecting the linear DNA molecule, the exonuclease is a bacterial exonuclease.
In another embodiment of the method of protecting the linear DNA molecule, the DNA molecule includes a functional DNA molecule.
In another embodiment of the method of protecting the linear DNA molecule, the DNA molecule includes a gene, a regulatory sequence, a splice site a binding site, a primer, an aptamer or combinations thereof.
In another embodiment of the method of protecting the linear DNA molecule, the DNA molecule includes a terminator sequence and the 3′ Ter site is downstream the terminator sequence.
In another embodiment of the method of protecting the linear DNA molecule, the DNA molecule includes a coding sequence for encoding an expression product and the terminator sequence is located after a STOP codon of the DNA molecule coding sequence and before the 3′ Ter.
In another embodiment of the method of protecting the linear DNA molecule, the method further comprises adding a 5′ DNA buffer region upstream the 5′ end of the DNA molecule and a 3′ DNA buffer region 3′ end downstream of the DNA molecule, and wherein the 5′ DNA buffer region includes between 0 to 300 base pairs and the 3′ DNA buffer region includes between 0 to 125 base pairs.
In another embodiment of the method of protecting the linear DNA molecule, the linear DNA molecule includes the coding sequence for encoding the expression product, and wherein the 3′ DNA buffer ranges between 45 and 125 base pairs.
In another embodiment of the method of protecting the linear DNA molecule, the Tus is provided as purified Tus or as a Tus-expressing bacterial strain.
In another embodiment of the method of protecting the linear DNA molecule, the Tus is provided as a Tus-expressing bacterial strain under control of an endogenous bacterial RNA polymerase.
In another embodiment of the method of protecting the linear DNA molecule, at least one of the one or more Ter sites comprises SEQ ID NO:1.
In another embodiment of the method of protecting the linear DNA molecule, at least one of the one or more Ter sites at the 5′ terminus comprises SEQ ID NO: 2 and the one or more Ter sites at the 3′ terminus comprises SEQ ID NO: 3.
In another embodiment, the present disclosure relates to a method of synthesizing a polypeptide of interest in a cell-free protein synthesis (CFPS) reaction mixture comprising: a) providing a linear dsDNA molecule of the present disclosure, wherein the DNA sequence of interest is a coding sequence for encoding the polypeptide of interest, b) providing a Tus protein, and c) adding the linear dsDNA and the Tus protein to the CFPS, thereby synthesizing the polypeptide of interest.
In one embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the 3′ Ter is downstream a terminator sequence.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the terminator sequence is located after a STOP codon of the DNA coding sequence and before the 3′ Ter.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the linear dsDNA molecule further comprises a 5′ DNA buffer region upstream the 5′ end of the DNA sequence of interest and a 3′ DNA buffer region 3′ end downstream the DNA sequence of interest, and wherein the 5′ DNA buffer region includes between 0 to 300 base pairs and the 3′ DNA buffer region includes between about 45 to about 125 base pairs.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the Tus protein is provided as a purified Tus protein.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the CFPS includes a bacteriophage RNA polymerase.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the Tus protein is provided as a Tus-expressing bacterial strain.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the CFPS is an E. coli lysate-based protein expression having endogenous E. coli RNA polymerase.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, the CFPS is derived from eukaryotes or prokaryotes.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, at least one of the one or more Ter sites comprises SEQ ID NO: 1.
In another embodiment of the method of synthesizing a polypeptide of interest in a CFPS reaction mixture, at least one of the one or more Ter sites at the 5′ terminus comprises SEQ ID NO: 2 and the one or more Ter sites at the 3′ terminus comprises SEQ ID NO: 3.
In another embodiment, the present disclosure provides for a cell transformed with a linear double stranded DNA according to any embodiment of the present disclosure. In one aspect, the cell is a bacterium.
In another embodiment, the present disclosure is a cell-free synthetic biology system comprising the linear dsDNA molecule as defined in any embodiment of the present invention.
In one embodiment, the cell-free synthetic biology system comprises an E. coli lysate, a V. natriegens lysate or a B. subtilis lysate.
In another embodiment of the cell-free synthetic biology system, the cell-free synthetic biology system is derived from eukaryotes or prokaryotes.
A detailed description of the preferred embodiments is provided herein below by way of example only and with reference to the following drawings, in which:
In the drawings, one embodiment of the disclosure is illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding and are not intended as a definition of the limits of the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.
All numerical designations, e.g., pH, temperature, time, concentration and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/−20%, +/−15%, or alternatively +/−10%, or alternatively +/−5% or alternatively +/−2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polypeptide” includes a plurality of polypeptides, including mixtures thereof.
As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
“Functional DNA Sequence” is meant to include a DNA sequence that is transcribed or bound by particular proteins or RNA molecules. Non-limiting examples of functional DNA sequences include a gene, a regulatory sequence, a splice site, a binding site, primers, aptamers and so forth.
A “terminator” is a DNA sequence-based element that defines the end of a transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription machinery.
A “cell free protein synthesis (CFPS)” reaction mixture typically contains a crude or partially-purified eukaryote or bacterial (such as E. coli, V. nat., S. subtillis) extract, a DNA or RNA translation template, and a suitable reaction buffer for promoting cell-free protein synthesis from the RNA translation template. In some aspects, the CFPS reaction mixture can include exogenous RNA translation template. In other aspects, the CFPS reaction mixture can include a DNA expression template encoding an open reading frame operably linked to a promoter element for a DNA-dependent RNA polymerase. In these other aspects, the CFPS reaction mixture can also include a DNA-dependent RNA polymerase to direct transcription of an RNA translation template encoding the open reading frame. In these other aspects, additional NTP's and divalent cation cofactor can be included in the CFPS reaction mixture. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of ordinary skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of ordinary skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components of the disclosure.
The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
The term “promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
Provided herein are linear double-stranded (ds) DNA sequences, molecules, constructs, systems and methods for linear, double-stranded DNA protection from exonuclease degradation (exonucleolytic degradation) that is highly efficient in cell free systems. The dsDNAs and methods of the present disclosure are based on the “Tus-Ter” DNA replication termination system found in bacteria, including homologues and variants of Tus and Ter across bacteria such as E. coli and many γ-proteobacterial strains [28]. The addition of the Ter sequence to linear expression templates (LETs) with free termini in the presence of Tus protein can provide potent protection of LETs from exonucleases in cell lysate-based expression systems.
The examples below show that one or more Ter sequences appended to the 5′ and 3′ ends of a linear DNA molecule in the presence of Tus, provides protection from exonuclease degradation in cell lysate-based expression systems (
The dsDNA sequences, molecules and/or constructs of the present disclosure can be used in any lysate, extract, system, cell-free system (CFS or CFSs for plural), etc., including patient sample lysates for diagnostics that includes or is suspected to include an exonuclease.
The use case of Tus-Ter constructs to protect DNA molecules from exonuclease degradation is not limited to protein expression. Tus-Ter can be used in other applications that involve linear DNA, such as signal amplification in diagnostics, biosensing gene circuits, or DNA sequencing; where Ter sites can be incorporated as primers to protect amplified DNA from exonucleolytic degradation in the in vitro enzymatic environment. Additionally, Tus-Ter can be used to protect pre-amplified functional DNA sequences such as aptamers, aptasensors and aptazymes in an in vitro environment. The Tus-Ter constructs described herein, can provide protein expression at levels similar to or higher than plasmid-based DNA inputs. Furthermore, Tus can be provided exogenously or endogenously expressed by recombinant expression; including for example under the control of the endogenous RNA Polymerase (RNAP). The Tus-Ter systems described herein are useful in CFSs derived from eukaryotes (e.g., vertebrates, plants, insects, fungi) or prokaryotes (e.g., Escherichia coli, Vibrio natriegens, Bacillus subtilis) and the CFSs may be prepared as either purified components or semi-processed cellular extracts. CFSs can be made sterile via simple filtration, which provides for a biosafe format for use outside of the lab.
The dsDNAs of the present disclosure have many applications, such as diagnostics, DNA amplification, DNA transcription, DNA translation, and so forth.
The following examples are intended to illustrate, but not limit the disclosure.
E. coli BL21 (C2530), BL21 (DE3) (C2527), 5-alafa (C2987), and SHuffle® Express (C3028) strains were purchased from NEB. V. nat (#14048) was purchased from ATCC. For plasmid construction, NEBuilder® HiFi DNA Assembly Master Mix (E2621) and standard molecular cloning procedures were used. pET24b-NusA-Tus, pET24b-mCherry and pET24b-deGFP were constructed based on the pET24b backbone from Addgene (#111702). pQE-PLlaco-T7 was constructed by replacing the T5 promoter in in pQE-T7911 (a kind gift from Prof Ben Luisi's laboratory (Cambridge, UK) and originally provided by Dr. Thomas Shrader (Albert Einstein College of Medicine, NY)) for PLlacO-1 promoter. For use as cell-free expression template, plasmids were propagated in 5-alfa cells and purified using E.Z.N.A.® Plasmid Midi Kit (D6904-03) from Omega Bio-Tek; and further concentrated using Amicon Ultra centrifugal filter units (Z648035) from Millipore Sigma. Plasmids were eluted in nuclease-free water and quantified on a Thermo Scientific™ NanoDrop™ One UV-Vis Spectrophotometer. In all cases, A260/280 and A260/A230 ratios were 1.8-1.85 and 2.1-2.3, respectively, indicating high purity. Additionally, agarose gel electrophoresis was used to confirm plasmid quality (
Q5® High-Fidelity DNA Polymerase (NEB M0491) was used for all PCRs. Primers were designed manually, checked on the SnapGene® Viewer Software, and synthesised by Eurofins Genomics or Integrated DNA Technologies. PCR reactions were all assembled in 100 μl volumes and contained 1×Q5® reaction buffer, 200 UM dNTPs, 500 nM each of forward and reverse primers, 1-10 ng of plasmid template, and 1 μl of Q5® Polymerase. PCRs were performed on an Applied Biosystems ProFlex™ thermocycler using the following conditions: 1 minute initial denaturation at 98° C.; followed by ×35 cycles of 6 second at 98° C., 15 seconds at 60° C., and 90 seconds at 72° C. After completion, all PCR products were subjected to DpnI (NEB R0176) digestion to ensure no plasmid template carryover. QIAquick PCR Purification Kit (Qiagen #28106) was used to purify DpnI digested PCR products, with final elution in 35 μl of nuclease-free water. PCR products' quantity and quality were checked as described above for plasmids, (see
E. coli based lysates were prepared essentially as described in Levine et al [32]. V. nat based lysates were prepared according to the guidelines set in Des Soyes et al [14]; and essentially following the protocols described in Levine et al with these modifications: Brain heart infusion (BHI) media containing v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14 mM MgCl2) was used for cell growth and cells were harvested at OD600 of 7.
Cell-free reactions' composition were based essentially on the protocols described previously with the following modifications: Phosphoenolpyruvate in the Solution B was replaced with 3-Phosphoglyceric acid, and Solution A did not contain putrescine; 20 amino acids were omitted from Solution B and added separately at a final concentration of 2.1 mM. The recipe for 20 amino acid (Sigma-Aldrich LAA21) stock solutions was adapted from with the following modifications: Arg was dissolved in ultra-pure water; and Asp, Glu, His and Tyr were dissolved in 3 M hydrochloric acid. The final concentration of T7 RNA polymerase and Tus protein were always set at 1.2 μM and 5 μM, respectively.
Purified Tus was not added to BL21-Tus lysate based reactions. Reactions with the endogenous E. coli RNAP were performed in Lysate A and supplemented with 0.05 units/μL of E. coli RNAP (NEB M0551). Where indicated, GamS (Arbor Biosciences #501024) was also added at 5 μM final concentration. Extract:total-reaction ratios were set as follows: 33% v/v for E. coli BL21, and 25% v/v for E. coli SHuffle® Express and V. nat. Cell-free reactions for each lysate were always assembled on ice as a master mix containing all components barring DNA templates, then thoroughly mixed and aliquoted so that the final volume of individual reactions was 20 μl after DNA addition. For plate reader measurements, each 20 μl reaction was immediately divided (on ice) into triplicate 6 μl volumes in a 386 Corning® microwell plate, sealed with a clear film (SARSTEDT 95.1994), and placed in a Synergy Neo2 plate reader (BioTek®). Reaction temperature was always set to 30° C., and fluorescence measurement settings were as follows: for mCherry, excitation at 587/10 nm and emission at 610/10 nm; and for deGFP, excitation at 488/9 nm and emission at 507/9 nm.
TUS: pET24b-NusA-Tus was transformed into BL21 (DE3) cells and used to inoculate an overnight Luria-Bertani (LB) culture growing at 37° C. The overnight culture was then diluted 1/200 into 1 L of fresh LB and incubated at 37° C. with shaking at 250 RPM until the cells reached mid-exponential phase (OD600=˜0.6). Tus expression was induced by the addition of 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown for an additional four hours at 37° C. with shaking at 250 RPM. Cells were then harvested by centrifugation at 8000 RCF for 15 minutes, saving the pellet. For protein purification, the cell pellet was resuspended in 20 ml of ion matrix affinity chromatography (IMAC) binding buffer (50 mM Tris-HCL (PH 7.8), 300 mM NaCl, one cOmplete™ EDTA-free Protease Inhibitor tablet) and subjected to sonication on a Fisherbrand™ Q700 sonicator with the following settings: 50% amplitude, 5 seconds ON for a total of 6 minutes with 10 second OFF cycles. The cell lysate was then centrifuged at 20,000 RCF for 1 hour at 4° C., and the supernatant was passed through a 0.2 μm Basix™ syringe filter. Using an ÄKTA Pure System (Cytiva), the cleared lysate was then passed through a 5 ml HisTrap FF IMAC column (Cytiva) and eluted in binding buffer containing 500 mM imidazole (without protease inhibitor tablet).
IMAC elution fractions were then pooled and subjected to TEV cleavage for 15 hours at room temperature to remove the NusA tag. The cleaved sample was then further purified using the ÄKTA Pure System on a HiLoad® 16/600 Superdex® 75 pg gel filtration column (Cytiva) equilibrated in Tus storage buffer (50 mM Tris-HCL (PH 7.8), 300 mM NaCl, 1 mM DTT). Final protein concentration was then determined using the molar extinction coefficient of Tus (ε=39420 M−1 cm−1) on a Thermo Scientific™ NanoDrop™ One UV-Vis Spectrophotometer, and aliquots were flash frozen and stored at −80° C.
T7 RNA polymerase: a similar protocol to Tus was used with the following changes: E. coli BL21 cells were used for protein expression, IMAC binding buffer contained 50 mM HEPES (PH 7.5), 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 20 mM imidazole, and one cOmplete™ EDTA-free Protease Inhibitor tablet; IMAC elution buffer was Binding buffer with 0.5 M imidazole and no protease inhibitor tablet; for gel filtration a HiLoad® 16/600 Superdex® 200 pg column equilibrated in T7 storage buffer (20 mM KH2PO4 (PH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.05% Triton X-100) was used; and the molar extinction coefficient of T7 RNA polymerase was (ε=140260 M−1 cm−1).
To demonstrate the effectiveness of the “Tus-Ter” protection strategy in CFSs we compared the expression levels of equimolar amounts of linear vs plasmid templates for mCherry and deGFP in E. coli and V. nat extracts. mCherry and deGFP have differing expression dynamics, with detectable signal appearing at approximately 70 and 12 minutes for mCherry (
For simple implementation, we included the purified Tus protein as an additive during reaction set up and incorporated the Ter sequence as a primer extension during PCR, without the need for Tus-LET preincubation. The addition of Tus or Ter per se does not have any detectable effect on the performance of CFSs (
Although the addition of defined amounts of purified Tus can allow for more control over individual experiments, using a Tus-expressing bacterial strain for lysate preparation can simplify the workflow in many CFS applications. We therefore tested the expression of selected LETs (TO and 0-125) in a Tus-expressing BL21-based lysate (BL21-Tus). As seen in
Cell-free gene expression under the control of endogenous RNA polymerases has been proven as a versatile tool for construction and characterization of synthetic gene circuits in recent years [8,36] due to the rich repertoire of endogenous transcription regulatory elements and the closer correlation between the in vitro and in vivo behavior of gene circuits under endogenous control. To demonstrate that the Tus-Ter system presented herein can confer nuclease protection on LETs under a sigma-70 promoter, we prepared linear templates based on the pBESTOR2—OR1-Pr-UTR1-deGFP-T500 plasmid. 34 Expectedly, overall expression rates were lower compared to the T7 RNAP-based system, and therefore protected linear versus plasmid expression levels were also lower (˜20%,
Remarkably, the Tus-Ter constructs and method of this disclosure were also capable of maintaining plasmid-level expression from linear templates in a V. nat CFS. However, here the effects of LET buffer region length as well as T7 terminator were much less pronounced. As seen in
GamS protein has often been used as a gold standard for comparing the LET protection efficiency of new methods in the field19,24,25. In order to see how Tus-Ter compares to GamS, we performed linear vs plasmid expression tests in cell-free reactions containing 5 μM of either Tus or GamS (
In terms of implementation, the Tus-Ter system/method presented herein is highly practicable and convenient-requiring minimal manipulations to the cell-free extracts or the linear templates. No strain engineering, or cumbersome post-PCR processing, or prohibitively long buffer regions are required. The Tus protein can be produced and purified from E. coli or other γ-proteobacterial strains in high quantities and added to cell-free reactions immediately before the addition of LETs. Likewise, the 23 bp Ter sequence can be conveniently added during commercial gene synthesis or as a primer overhang during PCR. Our results demonstrate the robust performance of Tus-Ter in two important chassis organisms, the established E. coli and the rapidly emerging V. nat. It has been further demonstrated that Tus-Ter protection of linear DNA can be achieved using endogenously expressed Tus including for example under the control of the endogenous E. coli RNA Polymerase (RNAP). We anticipate that Tus-Ter will be employed widely in research and commercial cell free applications for expedited discovery, especially when V. nat based CFSs are used.
Ter sequences are used as primer extensions in LAMP or RPA for isothermal amplification of low-abundance target pathogen sequences in the presence of Tus. For this, target-specific forward and reverse primers are synthesised with a 5′ Ter overhang. The concomitant binding of amplicons with Tus in the amplification reaction, or even the addition of Tus post-amplification, will result in added stability and therefore sensitivity in diagnostic and gene circuit-based assays; especially when these assays are performed under exonuclease-prone conditions. Such conditions may arise by the use of non-or-partially purified patient samples, or the use of crude enzyme mixtures, or potential residual exonuclease contamination during reaction set up. In all cases terminal blocking of target amplicons is likely to significantly increase their lifetime and thus boost the assays' sensitivity.
Pre-amplification of gene circuit, toehold or aptamer-based biosensing reporter sequences using Ter primers and binding with Tus prior to or during their addition to biological sample, for added stability and sensitivity. Here, toehold reporter or aptamer reporter-specific forward and reverse primers are synthesized with 5′ Ter overhangs and used for PCR amplification of target sequences. As above, if the assay environment is prone to exonuclease contamination such as that from crude enzyme solutions or unpurified biological samples, addition of Tus during or prior to the addition of Ter-reporter sequences can increase reporter stability and therefore assay sensitivity. Further, Tus-Ter can be used as a strand-clamp in gene circuit-based tools where spontaneous breathing or de-hybridization at termini may induce signal leakage, structure de-stabilisation or other failure modes.
Tus is immobilized on the surface of Lateral Flow diagnostic Assays to detect Ter-amplified target pathogen sequences. Here, isothermal amplification (LAMP or RPA) is performed on biological sample using target-specific forward and reverse primers containing functionally oriented Ter and reporter sequence (e.g. aptamer) overhangs. Also, following the general design principles of Lateral Flow Assays (LFAs), Tus is immobilized on two locations (Control and Test) on a standard LFA nitrocellulose strip. Followed by the Tus-Ter immobilization on the Control position of a pre-amplified control reporter construct containing both Ter and an e.g. reporter aptamer. The resulting isothermal amplification reaction solution can then be applied along with a reporter substrate to the Tus LFA strip. If the target pathogen sequence is present in the starting biological sample and successfully amplified, functional, double-stranded Ter and reporter sequences are reconstituted. As a result, the amplicons will be immobilized on the Test position on the LFA strip and the reporter sequences in both Control and test positions will react with the substrate. The user will then be able to detect the signal on each Control and Test position and make a judgement as to the presence or absence of pathogen in the starting biological sample.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050113 | 1/27/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63142097 | Jan 2021 | US |
