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Evolution made easy
Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.
Eugene J. H. Wee and Matt Trau
C
hemists, biologists and engineers have been developing high-throughput screening (HTS) strategies for synthetic molecules since the invention of combinatorial chemistry by Geysen1 and Nielsen2 during the 1980s and early 1990s. All of these approaches have a similar theme: (i) a randomized chemical library, (ii) a molecular ‘bait-and-hook’ selection strategy and (iii) an engineered screening method to select for ‘hits’. As well as a plethora of chemical systems currently available, biological systems, for example, yeast 3 and phage4 display, have also been co-opted and engineered for HTS selection of protein binding molecules for a diverse array of applications including drug leads, novel enzymes and agents for diagnostic assays. A popular HTS strategy is the in vitro compartmentalization (IVC) method5. This relies on the formation of emulsion droplets in microfluidic systems that capture molecular substrates so that each droplet is effectively a single reaction capsule. The droplet formation can be tuned — by adjusting the flow rates of the emulsion components — so that a substrate can be encased with a single molecule of enzyme in a single droplet (the content of droplets follows a Poisson distribution). Screening for enzyme activity can then be performed using modified fluorescence-activated cell sorting (FACS) that can enable ultra-high-throughput screening (UHTS). One of the attractive benefits of IVC is the potential ability to couple genotype and phenotype — that is, to maintain the connection between the genetic content (DNA sequence) and the observable characteristics (for example, enzyme activity). This is, however, a major limitation of emulsion droplet systems because once the emulsion is broken it is difficult to track genotypes. Various labelled DNA, affinity capture or proxy barcoding strategies have been developed to circumvent this limitation6–9. Briefly, these strategies are secondary ‘selection’ methods that indirectly report on the genotype of the newly evolved protein. 756
Expression in E. coli
6
1
Droplet formation, cell lysis
Genotype recovery
2
5 Catalysis Selection by FACS
4
Plasmid
Gel-shell bead formation
Enzyme
Substrate (with tag)
3
Product (with tag)
Figure 1 | Directed evolution of enzymes in gel-shell beads10. A library of enzymes is created by expression within Escherichia coli carrying the encoding plasmid DNA. The bacteria are encapsulated in droplets so that each droplet contains a single enzyme and the DNA that encodes it. Cell lysis releases the enzyme so that it can be tested as a catalyst with the activity measured using fluorescence. Gel-shell bead formation traps the enzyme with its encoding DNA and the fluorescent reaction product so that active enzymes can be identified in a high-throughput fashion for analysis of the DNA sequence, and potentially for further rounds of enzyme evolution.
Now, writing in Nature Chemistry, Florian Hollfelder and co-workers have described10 a convenient and low-cost approach that allows simultaneous purification of both an enzyme and the DNA that encodes it. They use this system to perform a high-throughput evolution of a phosphotriesterase enzyme that can be used in the detoxification of pesticides or nerve agents. Their “allin-one” strategy (Fig. 1) relies on the generation of “biomimetic gel-shell beads” for IVC. In a similar fashion to other droplet formation/FACS strategies, their method first generates emulsion droplets so that each droplet encapsulates a single
bacterium carrying a variant of the enzyme (at both the protein and DNA levels), enzyme substrate, low-melting agarose, lysis buffer and an alginate polyanion. Lysis of the trapped bacteria followed by solidification of the agarose in the presence of poly(allylamine-hydrochloride) results in the formation of a porous agarose gelbead containing the released enzyme, the encoding DNA and the enzyme substrate. Thus both the phenotype and encoding genotype (an expression plasmid) are trapped in a convenient bead-like substrate amenable to a standard FACS process. After identifying beads that contain active enzymes, the encoding DNA can
NATURE CHEMISTRY | VOL 6 | SEPTEMBER 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
news & views be recovered for analysis and potentially further rounds of evolution. Like many other UHTS strategies, the demonstrated application of this UHTS strategy is for directed evolution of enzymes, but one could imagine other applications. For example, there is a profound need for methods to screen and validate the vast number of potential cancer biomarkers being discovered11. However, the expression of functional full-length mammalian proteins on a scale suitable for current biochemical assays is a major challenge. A UHTS approach such as this one, enabled for mammalian protein expression, could benefit fundamental cancer biology, drug design and diagnostics. Another plausible application could be in the emerging field of designer enzymes. For example, one may consider using a combinatorial chemistry approach to combine different enzymatic domains to generate new enzymes with the combined functionality of the parent enzymes. UHTS approaches using materials that are compatible with cell-free protein expression12 or the gel-shell bead method described by Hollfelder and co-workers could facilitate such an endeavour. One major limitation of FACS-based assays is the requirement for a fluorescent reaction product. A possible solution could be the use of a secondary enzyme cascade to generate a fluorescent signal. Another prospective avenue UHTS methods can adopt is to assimilate with classical
protein/protein screening strategies such as yeast and phage display systems (mentioned above). A potential immediate application would combine the speed of yeast-expressed antibody fragment systems13 with UHTS methods such as Hollfelder’s to further enhance in vitro evolution of antibody vaccines against new strains of infectious pathogens (for example the flu virus). In vitro biological HTS methods have come a long way from the classical 96-wellplate formats. However, the adoption of such new methods in practice can be slow (as with any new methodology). The main barrier to entry is likely to be the need for proprietary equipment, reagents and protocols. For example, the crucial step in IVC is the generation of emulsion droplets, which requires precise control to generate monodisperse droplets. In an emerging variant of the polymerase chain reaction (PCR) called digital PCR, limiting dilutions are achieved with either emulsion droplets similar to that described for IVC, or on a solid platform chip with tens of thousands of chemically coated wells14. The latter has significantly easier protocols for generating a Poisson distribution of analytes without the need to generate monodispersed emulsion droplets. Hence it seems highly likely that future iterations of IVC might evolve onto more convenient platforms before becoming a mainstream tool. In conclusion, there is a still a need to further simplify, miniaturize and adapt
current protein UHTS approaches for routine practice. Simple all-in-one UTHS solutions would benefit not only general proteomics but also drug design and cancer diagnostics in a similar fashion to how DNA sequencing technology has evolved into convenient, low-cost bench-top applications to revolutionize modern genomics. ❐ Eugene Wee and Matt Trau are at the Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Brisbane, Queensland, 4072, Australia. Matt Trau is also at the School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, 4072, Australia. e-mail: [email protected] References
1. Geysen, H. M., Meloen, R. H. & Barteling, S. J. Proc. Natl Acad. Sci. USA 81, 3998–4002 (1984). 2. Nielsen, J., Brenner, S. & Janda, K. D. J. Am. Chem. Soc. 115, 9812–9813 (1993). 3. Boder, E. T. & Wittrup, K. D. Nature Biotechnol. 15, 553–557 (1997). 4. Smith, G. P. Science 228, 1315–1317 (1985). 5. Tawfik, D. S. & Griffiths, A. D. Nature Biotechnol. 16, 652–656 (1998). 6. Yonezawa, M., Doi, N., Higashinakagawa, T. & Yanagawa, H. J. Biochem. 135, 285–288 (2004). 7. Bertschinger, J. & Neri, D. Protein Eng. Des. Sel. 17, 699–707 (2004). 8. Figueiredo, P., Roberts, R. L. & Nester, E. W. Proteomics 4, 3128–3140 (2004). 9. Nord, O., Uhlén, M. & Nygren, P.-Å. J.Biotechnol. 106, 1–13 (2003). 10. Fischlechner, M. et al. Nature Chem. 6, 791–796 (2014). 11. Hartwell, L., Mankoff, D., Paulovich, A., Ramsey, S. & Swisher, E. Nature Biotechnol. 24, 905–908 (2006). 12. Mureev, S., Kovtun, O., Nguyen, U. T. T. & Alexandrov, K. Nature Biotechnol. 27, 747–752 (2009). 13. Holliger, P. & Hudson, P. J. Nature Biotechnol. 23, 1126–1136 (2005). 14. Baker, M. Nature Methods 9, 541–544 (2012).
TWO-DIMENSIONAL MATERIALS
Crystallized creations in 2D
Two reports demonstrate that with the right molecules and the right crystalline arrangement, it is not only possible to create two-dimensional crystals, but also to separate them into single-molecule-thick sheets — so-called two-dimensional polymers.
Neil R. Champness
T
he discovery of graphene and its remarkable properties has rejuvenated interest in developing bespoke two-dimensional materials — that is, atomically thin materials with periodicity in the other two spatial directions. Creating two-dimensional molecular arrays with specific physical and chemical properties is an alluring goal, and such structures have potential in a variety of applications. For example, consider a molecular-scale membrane comprising highly organized components with specific properties — be they optical, magnetic or
electronic — and the ability to recognize specific target molecules through the incorporation of tailored recognition sites. Sounds like a chemist’s dream! Clearly, these materials are highly attractive targets, but such systems are also exceedingly difficult to obtain using current synthetic methods due to a lack of control over directionality of intermolecular reactions. Now, as they describe in two separate Articles in Nature Chemistry, Benjamin King 1 and Dieter Schlüter 2 and their respective co-workers have taken great strides forward in addressing these
NATURE CHEMISTRY | VOL 6 | SEPTEMBER 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
challenges by utilizing highly ordered crystalline structures to prearrange components of the molecular arrays. A number of strategies have been employed over recent years to create periodic two-dimensional arrays; these often include using surfaces as templates for covalent 3 or supramolecular arrays4. However, such approaches cannot typically form samples with both longrange order and extended dimensions, and they can also be complicated by far from innocent surfaces. The approach employed by both King and Schlüter is to 757
Evolution made easy
Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format.
Eugene J. H. Wee and Matt Trau
C
hemists, biologists and engineers have been developing high-throughput screening (HTS) strategies for synthetic molecules since the invention of combinatorial chemistry by Geysen1 and Nielsen2 during the 1980s and early 1990s. All of these approaches have a similar theme: (i) a randomized chemical library, (ii) a molecular ‘bait-and-hook’ selection strategy and (iii) an engineered screening method to select for ‘hits’. As well as a plethora of chemical systems currently available, biological systems, for example, yeast 3 and phage4 display, have also been co-opted and engineered for HTS selection of protein binding molecules for a diverse array of applications including drug leads, novel enzymes and agents for diagnostic assays. A popular HTS strategy is the in vitro compartmentalization (IVC) method5. This relies on the formation of emulsion droplets in microfluidic systems that capture molecular substrates so that each droplet is effectively a single reaction capsule. The droplet formation can be tuned — by adjusting the flow rates of the emulsion components — so that a substrate can be encased with a single molecule of enzyme in a single droplet (the content of droplets follows a Poisson distribution). Screening for enzyme activity can then be performed using modified fluorescence-activated cell sorting (FACS) that can enable ultra-high-throughput screening (UHTS). One of the attractive benefits of IVC is the potential ability to couple genotype and phenotype — that is, to maintain the connection between the genetic content (DNA sequence) and the observable characteristics (for example, enzyme activity). This is, however, a major limitation of emulsion droplet systems because once the emulsion is broken it is difficult to track genotypes. Various labelled DNA, affinity capture or proxy barcoding strategies have been developed to circumvent this limitation6–9. Briefly, these strategies are secondary ‘selection’ methods that indirectly report on the genotype of the newly evolved protein. 756
Expression in E. coli
6
1
Droplet formation, cell lysis
Genotype recovery
2
5 Catalysis Selection by FACS
4
Plasmid
Gel-shell bead formation
Enzyme
Substrate (with tag)
3
Product (with tag)
Figure 1 | Directed evolution of enzymes in gel-shell beads10. A library of enzymes is created by expression within Escherichia coli carrying the encoding plasmid DNA. The bacteria are encapsulated in droplets so that each droplet contains a single enzyme and the DNA that encodes it. Cell lysis releases the enzyme so that it can be tested as a catalyst with the activity measured using fluorescence. Gel-shell bead formation traps the enzyme with its encoding DNA and the fluorescent reaction product so that active enzymes can be identified in a high-throughput fashion for analysis of the DNA sequence, and potentially for further rounds of enzyme evolution.
Now, writing in Nature Chemistry, Florian Hollfelder and co-workers have described10 a convenient and low-cost approach that allows simultaneous purification of both an enzyme and the DNA that encodes it. They use this system to perform a high-throughput evolution of a phosphotriesterase enzyme that can be used in the detoxification of pesticides or nerve agents. Their “allin-one” strategy (Fig. 1) relies on the generation of “biomimetic gel-shell beads” for IVC. In a similar fashion to other droplet formation/FACS strategies, their method first generates emulsion droplets so that each droplet encapsulates a single
bacterium carrying a variant of the enzyme (at both the protein and DNA levels), enzyme substrate, low-melting agarose, lysis buffer and an alginate polyanion. Lysis of the trapped bacteria followed by solidification of the agarose in the presence of poly(allylamine-hydrochloride) results in the formation of a porous agarose gelbead containing the released enzyme, the encoding DNA and the enzyme substrate. Thus both the phenotype and encoding genotype (an expression plasmid) are trapped in a convenient bead-like substrate amenable to a standard FACS process. After identifying beads that contain active enzymes, the encoding DNA can
NATURE CHEMISTRY | VOL 6 | SEPTEMBER 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
news & views be recovered for analysis and potentially further rounds of evolution. Like many other UHTS strategies, the demonstrated application of this UHTS strategy is for directed evolution of enzymes, but one could imagine other applications. For example, there is a profound need for methods to screen and validate the vast number of potential cancer biomarkers being discovered11. However, the expression of functional full-length mammalian proteins on a scale suitable for current biochemical assays is a major challenge. A UHTS approach such as this one, enabled for mammalian protein expression, could benefit fundamental cancer biology, drug design and diagnostics. Another plausible application could be in the emerging field of designer enzymes. For example, one may consider using a combinatorial chemistry approach to combine different enzymatic domains to generate new enzymes with the combined functionality of the parent enzymes. UHTS approaches using materials that are compatible with cell-free protein expression12 or the gel-shell bead method described by Hollfelder and co-workers could facilitate such an endeavour. One major limitation of FACS-based assays is the requirement for a fluorescent reaction product. A possible solution could be the use of a secondary enzyme cascade to generate a fluorescent signal. Another prospective avenue UHTS methods can adopt is to assimilate with classical
protein/protein screening strategies such as yeast and phage display systems (mentioned above). A potential immediate application would combine the speed of yeast-expressed antibody fragment systems13 with UHTS methods such as Hollfelder’s to further enhance in vitro evolution of antibody vaccines against new strains of infectious pathogens (for example the flu virus). In vitro biological HTS methods have come a long way from the classical 96-wellplate formats. However, the adoption of such new methods in practice can be slow (as with any new methodology). The main barrier to entry is likely to be the need for proprietary equipment, reagents and protocols. For example, the crucial step in IVC is the generation of emulsion droplets, which requires precise control to generate monodisperse droplets. In an emerging variant of the polymerase chain reaction (PCR) called digital PCR, limiting dilutions are achieved with either emulsion droplets similar to that described for IVC, or on a solid platform chip with tens of thousands of chemically coated wells14. The latter has significantly easier protocols for generating a Poisson distribution of analytes without the need to generate monodispersed emulsion droplets. Hence it seems highly likely that future iterations of IVC might evolve onto more convenient platforms before becoming a mainstream tool. In conclusion, there is a still a need to further simplify, miniaturize and adapt
current protein UHTS approaches for routine practice. Simple all-in-one UTHS solutions would benefit not only general proteomics but also drug design and cancer diagnostics in a similar fashion to how DNA sequencing technology has evolved into convenient, low-cost bench-top applications to revolutionize modern genomics. ❐ Eugene Wee and Matt Trau are at the Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Brisbane, Queensland, 4072, Australia. Matt Trau is also at the School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, 4072, Australia. e-mail: [email protected] References
1. Geysen, H. M., Meloen, R. H. & Barteling, S. J. Proc. Natl Acad. Sci. USA 81, 3998–4002 (1984). 2. Nielsen, J., Brenner, S. & Janda, K. D. J. Am. Chem. Soc. 115, 9812–9813 (1993). 3. Boder, E. T. & Wittrup, K. D. Nature Biotechnol. 15, 553–557 (1997). 4. Smith, G. P. Science 228, 1315–1317 (1985). 5. Tawfik, D. S. & Griffiths, A. D. Nature Biotechnol. 16, 652–656 (1998). 6. Yonezawa, M., Doi, N., Higashinakagawa, T. & Yanagawa, H. J. Biochem. 135, 285–288 (2004). 7. Bertschinger, J. & Neri, D. Protein Eng. Des. Sel. 17, 699–707 (2004). 8. Figueiredo, P., Roberts, R. L. & Nester, E. W. Proteomics 4, 3128–3140 (2004). 9. Nord, O., Uhlén, M. & Nygren, P.-Å. J.Biotechnol. 106, 1–13 (2003). 10. Fischlechner, M. et al. Nature Chem. 6, 791–796 (2014). 11. Hartwell, L., Mankoff, D., Paulovich, A., Ramsey, S. & Swisher, E. Nature Biotechnol. 24, 905–908 (2006). 12. Mureev, S., Kovtun, O., Nguyen, U. T. T. & Alexandrov, K. Nature Biotechnol. 27, 747–752 (2009). 13. Holliger, P. & Hudson, P. J. Nature Biotechnol. 23, 1126–1136 (2005). 14. Baker, M. Nature Methods 9, 541–544 (2012).
TWO-DIMENSIONAL MATERIALS
Crystallized creations in 2D
Two reports demonstrate that with the right molecules and the right crystalline arrangement, it is not only possible to create two-dimensional crystals, but also to separate them into single-molecule-thick sheets — so-called two-dimensional polymers.
Neil R. Champness
T
he discovery of graphene and its remarkable properties has rejuvenated interest in developing bespoke two-dimensional materials — that is, atomically thin materials with periodicity in the other two spatial directions. Creating two-dimensional molecular arrays with specific physical and chemical properties is an alluring goal, and such structures have potential in a variety of applications. For example, consider a molecular-scale membrane comprising highly organized components with specific properties — be they optical, magnetic or
electronic — and the ability to recognize specific target molecules through the incorporation of tailored recognition sites. Sounds like a chemist’s dream! Clearly, these materials are highly attractive targets, but such systems are also exceedingly difficult to obtain using current synthetic methods due to a lack of control over directionality of intermolecular reactions. Now, as they describe in two separate Articles in Nature Chemistry, Benjamin King 1 and Dieter Schlüter 2 and their respective co-workers have taken great strides forward in addressing these
NATURE CHEMISTRY | VOL 6 | SEPTEMBER 2014 | www.nature.com/naturechemistry
© 2014 Macmillan Publishers Limited. All rights reserved
challenges by utilizing highly ordered crystalline structures to prearrange components of the molecular arrays. A number of strategies have been employed over recent years to create periodic two-dimensional arrays; these often include using surfaces as templates for covalent 3 or supramolecular arrays4. However, such approaches cannot typically form samples with both longrange order and extended dimensions, and they can also be complicated by far from innocent surfaces. The approach employed by both King and Schlüter is to 757
