Publications

3. Cotranscriptional RNA strand displacement underlies the regulatory function of the E. coli thiB TPP translational riboswitch.
Katherine E. Berman, Russell Steans, Laura M. Hertz, and Julius B. Lucks. (2022). doi: 10.1101/2022.08.24.505126
Links: BioRxiv

This paper we investigate the role of cotranscriptional RNA strand displacement on the E. coli thiB TPP riboswitch.  Previous work has demonstrated the importance of strand displacement in the switching mechanism of transcriptional riboswitches, but here we apply this hypothesis to a translational riboswitch using cellular fluorescence assays.  Our results suggest that an intermediate structure can strand displace the P1 helix allowing the riboswitch to switch between ribosome binding site sequestering and anti-sequestering stem.

2. De-novo design of translational RNA repressors.
P. D. Carlson, C. J. Glasscock, J. B. Lucks*. (2018). doi: 10.1101/501767
Links: BioRxiv

In this study, we developed two new classes of RNA regulators: (1) toehold repressors and (2) looped antisense oligonucleotides (LASOs) in an effort to elucidate design principles for repressive RNA interactions. We demonstrated that these RNAs efficiency repress translation of a downstream gene in response to an input RNA, are highly orthogonal and can be multiplexed with translational activators. Finally, we used LASO design to repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity.

1. Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations.
A. M Yu, M. E. Evans, J. B. Lucks. (2018) doi: 10.1101/292532
Links: BioRxiv

In this work, we present a derivation of RNA chemical probing reactivity calculations that includes signal from both reverse transcriptase stops and mutations. Intriguingly, the reactivity at each nucleotide is the sum of the stop signal and the mutation signal due to the mutual exclusivity of stops and mutations in a single cDNA.

76. Tuning strand displacement kinetics enables programmable ZTP riboswitch dynamic range in vivo.
D. Z. Bushhouse, J. B. Lucks. Nucleic Acids Research (2023). doi: 10.1093/nar/gkad110
Links: Journal

75. The accuracy and usability of point-of-use fluoride biosensors in rural Kenya.
W. Thavarajah, P. M. Owuor, D. R. Awuor, K. Kiprotich, R. Aggarwal, J. B. Lucks, S. L. Young. NPJ Clean Water (2023). doi: 10.1038/s41545-023-00221-5
Links: Journal

74. Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles.
M. A. Boyd, W. Thavarajah, J. B. Lucks, N. P. Kamat. Science Advances (2023). doi: 10.1126/sciadv.add6605
Links: Journal

73. Zur and Zinc Increase Expression of E. coli Ribosomal Protein L31 Through RNA-Mediated Repression of the Repressor L31p.
R. A. Rasmussen, S. Wang, J. M. Camarillo, V. Sosnowski, B-K. Cho, Y. A. Goo, J. B. Lucks, T. V. O’Halloran. Nucleic Acids Research (2022). doi: 10.1101/2022.05.27.493739
Links: Journal, PDF

We elucidate a new mechanism in this work that explains how E. coli switch one of their ribosomal proteins to adapt to the environmental stress zinc depletion. We find that one form of this ribosomal protein, which is regulated by a zinc-sensing transcription factor, represses production of the other protein by binding to its mRNA in a structure-dependent manner.

72. Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing
Harbaugh S. V., Silverman A. D., Chushak Y. G., Zimlich K., Wolfe M., Thavarajah W., Jewett M. C., Lucks J. B., Chávez J. L. ACS Synthetic Biology (2022). doi:10.1021/acssynbio.1c00560
Links: Journal, PDF

This paper uses RNA engineering to create a synthetic RNA riboswitch to detect dopamine in cell-free diagnostic systems. Use of control reactions allows an assay format to control for matrix inhibition when testing human samples.

71. How does RNA fold dynamically?
Bushhouse, D.Z., Choi, E.K., Hertz, L.M. and Lucks, J.B.*, Journal of Molecular Biology (2022) doi:10.1016/j.jmb.2022.167665
Links: Journal, PDF

In this perspective, we engage three questions regarding RNA cotranscriptional folding:
1) What is the Appropriate Biophysical Framework to Describe Non- equilibrium Conformational Switching?
2) How Widespread is Strand Displacement as a General Feature of RNA Conformational Switching?
3) How do Proteins Mediate Cotranscriptional RNA Conformational Switching?

70. Programming cell-free biosensors with DNA strand displacement circuits
K. J. Jung#, K. K. Alam#, Chloé M. Archuleta, J. B. Lucks*. Nature Chemical Biology (2022). doi:10.1038/s41589-021-00962-9. News and Views: “Logic invades cell-free biosensing.” E. Amalfatino and K. Pardee, Nature Chemical Biology, doi:10.1038/s41589-021-00963-8.
Links: Journal

Here we extend the ROSALIND cell-free biosensor platform with toe-hold mediated DNA strand displacement (TMSD) circuits. TMSD produces rapid signal generation, and provides a powerful approach to creating information processing circuits that improve and enhance biosensor function. We use TMSD to build 12 different logic gates that implement a range of functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND), as well as an analog-to-digital conversion circuit that gives semi-quantitative read outs.

69. Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch
L. Cheng, E. N. White, N. L. Brandt, A. M. Yu, A. A. Chen, J. B. Lucks. Nucleic Acids Research (2022). doi: 10.1101/2021.10.25.465737
Links: BioRxiv, Journal

Our work in this paper studies how RNA cotranscriptional folding is involved in the mechanism of a transcriptional purine-sensing riboswitch. We used a combination of SHAPE-Seq structural probing, cellular fluorescence assays, and all-atom molecular dynamics simulations that suggested an intermediate structure undergoes strand exchange to mediate between the anti-terminated and terminated states of the riboswitch.

68. Programming cell-free biosensors with DNA strand displacement circuits.
K. J. Jung#, K. K. Alam#, C. M. Archuleta, J. B. Lucks*. (2022). Nature Chemical Biology (2022). doi:10.1038/s41589-021-00962-9.
Link: Journal

News and Views: “Logic invades cell-free biosensing.” E. Amalfatino and K. Pardee, Nature Chemical Biology, doi:10.1038/s41589-021-00963-8.

67. ROSALIND: Rapid Detection of Chemical Contaminants with In Vitro Transcription Factor-Based Biosensors
J. K. Jung, K. K. Alam, J.B. Lucks*. Cell-Free Gene Expression, pp. 325-342 (2022).
Links: Book

This paper outlines detailed protocols for assembling ROSALIND cell-free biosensing reactions. Includes protocols for making reagents (DNA templates, purified transcription factors) as well.

66. RNA Engineering for Public Health: Innovations in RNA-Based Diagnostics and Therapeutics.
W. Thavarajah#, L. M. Hertz#, D. Z. Bushhouse, C. M. Archuleta, and J.B. Lucks*. Annual Reviews (2021). doi: 10.1146/annurev-chembioeng-101420-014055, # = Equal contribution
Links: Journal, PDF

This review describes recent applications in RNA engineering to develop the next generation of diagnostics and therapeutics. It covers sensors for both chemicals and pathogens, CRISPR technologies, mRNA vaccines, and the future of personalized medicine with RNA-targeting drugs. We wrap up with a perspective on the future of the field with its coming challenges and opportunities.

65. Dynamic control of gene expression with riboregulated switchable feedback promoters.
C.J. Glasscock, J.T. Lazar, B.W. Biggs, J.H. Arnold, M-K Kang, D. Tullman-Ercek, K.E.J. Tyo, J.B. Lucks*. ACS Synthetic Biology (2021). doi: 10.1021/acssynbio.1c00015
Links: Journal, BioRxiv, PDF

This paper presents a new mechanism for controlling cellular stress response networks with RNA regulators. The insertion of a small transcription activating RNA switch behind stress response promoters allows optimization of metabolic pathway production via induction timing, magnitude and even autonomous quorum sensing-mediated activation.

64. RNA sequence and structure determinants of Pol III transcriptional termination in human cells
Matthew S. Verosloff, William K. Corcoran, Taylor B. Dolberg, Joshua N. Leonard*, J.B. Lucks*. Journal of Molecular Biology (2021). doi: 10.1016/j.jmb.2021.166978
Links: Journal, BioRxiv , PDF

Here, we present work aimed at furthering our understanding of human RNA Polymerase III termination. To accomplish this, we developed a novel in celluo assay that allows for quantification of termination events. We demonstrated that in our system termination only occurs when poly-U tracts are longer than the average lengths found within the human genome. We found that placing secondary structure, immediately upstream of these tracts, within the nascent RNA enabled efficient termination at shorter but representative poly-U tract lengths. This impact decreases as we moved the structural motif further upstream from the poly-U tract.

63. Computationally reconstructing cotranscriptional folding pathways from experimental data reveals rearrangement of non-native folding intermediates.
A. M. Yu, P. M. Gasper, L. Cheng, L. B. Lai#, S. Kaur#, V. Gopalan, A. A. Chen, J. B. Lucks*. Molecular Cell (2021) doi: 10.1016/j.molcel.2020.12.017 # = Equal contribution
Links: Journal, BioRxiv, PDF

This paper presents a new method – reconstructing RNA dynamics from data (R2D2) – that combines high throughput chemical probing of RNA structure with computational structure prediction, to reconstruct cotranscriptional folding pathways from RNAs. Integration with all-atom 3D simulations allows mechanistic investigation into cotranscriptional RNA structure rearrangements. Application to the bacterial signal recognition particle (SRP) RNA strongly suggests a role for toehold mediated strand displacement in resolving kinetic traps in cotranscriptional folding pathways.

62. Cell-free biosensors for rapid detection of water contaminants.
J. K. Jung#, K. K. Alam#, M. S. Verosloff, D. A. Capdevila, M. Desmau, P. R. Clauer, J. W. Lee, P. Q. Nguyen, P. A. Pasten, S. J. Matiasek, J.-F. Gaillard, D. P. Giedroc, J. J. Collins, J. B. Lucks*. Nature Biotechnology (2020) doi: 10.1038/s41587-020-0571-7 # = Equal contribution
Links: Journal, BioRxiv, PDF

We developed a novel biosensing technique for detecting chemical contaminants in water using only transcription reactions. This platform called ROSALIND (RNA Output Sensors Activated by Ligand Induction) combines RNA polymerases, allosteric transcription factors, and synthetic DNA templates to regulate the synthesis of a fluorescent RNA aptamer. We applied ROSALIND to detect antibiotics, small molecules, and metals and took it out to the field to test municipal water supplies. RNA circuits are used to fix issues with transcription factor cross-talk and sensitivity with no protein engineering required.

61. Chemical transcription roadblocking for nascent RNA display
Eric J. Strobel, John T. Lis, Julius B. Lucks. Journal of Biological Chemistry.(2020) doi: 10.1101/779827
Links: Journal, BioRxiv, PDF

Here we present a new method for stalling transcription complexes using chemically modified DNA transcription templates. Templates can be generated by using primer extension reactions that makes them inexpensive to use and amenable to multiplexing.

60. A primer on emerging field-deployable synthetic biology tools for global water quality monitoring
Walter Thavarajah, Matthew S. Verosloff, Jaeyoung K. Jung, Khalid K. Alam, Joshua D. Miller, Michael C. Jewett, Sera L. Young, Julius B. Lucks . npj Clean Water.(2020) doi: 10.1038/s41545-020-0064-8
Links:Journal

This primer describes a suite of new synthetic biology technologies for low-cost, rapid and easy-to-use water quality monitoring. After a brief overview of biosensors, the primer goes into emerging technologies to detect pathogens and chemical contaminants. The piece ends with a forward looking perspective on what is needed to get these technologies out into the world.

59. Design and optimization of a cell-free atrazine biosensor.
A.D. Silverman, U. Akova, K.K. Alam, M.C. Jewett*, J. B. Lucks*. ACS Synthetic Biology.(2020) doi: 10.1021/acssynbio.9b00388
Links: Journal, BioRxiv, PDF

This paper details a mechanism for cell-free “metabolic biosensing” using the test case of detecting atrazine, which has no natural sensor, by supplementing a cell-free reaction with exogenous enzymes that convert atrazine to cyanuric acid, which is detectable. Since each pathway enzyme is pre-enriched in a separate E. coli extract, the sensor can be linearly tuned with ease.

58. Point-of-use detection of environmental fluoride via a cell-free riboswitch-based biosensor.
W. Thavarajah, A. D. Silverman, M. S. Verosloff, N. Kelley-Loughnane, M. C. Jewett, J. B. Lucks*. ACS Synthetic Biology.(2020) doi: 10.1021/acssynbio.9b00348.
Links: Journal, BioRxiv, PDF

In this work, we apply our fundamental knowledge of RNA sensing mechanisms to meet a global health challenge. By using a fluoride riboswitch in a freeze-dried cell-free reaction, we have created a powerful tool for point-of-use detection of a high-impact environmental contaminant.

57. Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid.
X. Liu, A. D. Silverman, K. K. Alam, E. Iverson, J. B. Lucks*, M. C. Jewett*, S. Raman*. ACS Synthetic Biology. (2020) doi: 10.1021/acssynbio.9b00348.
Links: Journal, BioRxiv, PDF

This collaborative paper with the Raman lab details the parallel construction of a cellular and cell-free biosensor for cyanuric acid, a model analyte in the broad LysR-type transcriptional regulator (LTTR) family.

56. De novo-designed translational repressors for multi-input cellular logic.
J. Kim, Y. Zhou, P. D. Carlson, M. Teichmann, F. C. Simmel, P. A. Silver, J. J. Collins, J. B. Lucks, P. Yin, A. A. Green*. Nature Chemical Biology. (2019). doi: 10.1101/501783
Links: Journal, BioRxiv, PDF

In this study, we developed two new classes of RNA regulators: (1) toehold repressors and (2) looped antisense oligonucleotides (LASOs) in an effort to elucidate design principles for repressive RNA interactions. We demonstrated that these RNAs efficiency repress translation of a downstream gene in response to an input RNA, are highly orthogonal and can be multiplexed with translational activators. Finally, we used LASO design to repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity.

55. DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data.
A. Y Xu, A. M Yu, J. B. Lucks, N. Bagheri.* Bioinformatics. (2019). doi: 10.1093/bioinformatics/btz449.
Links: Journal, PDF, Blog Post

Here we present a method to find reactivity patterns within cotranscriptional SHAPE-Seq datasets in an automated fashion without human intervention. Patterns of increasing or decreasing reactivities in specific nucleotides across different transcript lengths reveal cotranscriptional RNA folding events.

54. A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control.
E. J. Strobel*, L. Cheng, K. E. Berman, P. D. Carlson, J. B. Lucks*. Nature Chemical Biology. (2019). doi: 10.1101/521930
Links: Journal, BioRxiv,PDF, News & Views, SHAPE-Seq Datasets, RMDB, Sequence Read Archive , Source Data Files

Here we use cotranscriptional SHAPE-Seq, and a newly developed high throughput sequencing-based transcriptional assay to dissect the mechanism of folding of the ZTP riboswitch. We find that strand displacement in a critical region of the riboswitch expression platform governs the transcriptional regulatory decision, and uncover sequence determinants of efficient strand displacement.

53. Computational design of three-dimensional RNA structure and function.
J. D. Yesselman, D. Eiler, E. D. Carlson, M. R. Gotrik, A. E. d’Aquino, A. N. Ooms, W. Kladawang, P. D. Carlson, X. Shi, D. A. Constantino, D. Herschlag, J. B. Lucks, M. C. Jewett, J. S. Keift, R. Das. Nature Nanotechnology. (2019). doi: 10.1038/s41565-019-0517-8.
Links: Journal, PDF

This paper describes RNA-Make, a computational tool that can design dimensional RNA structures. RNA-Make is used to design tethers for tethered ribosomes and stabilizing scaffolds for fluorescent RNA aptamers. RNA-Make opens up a new capability in computational RNA design.

52. Tracking RNA structures as RNAs transit through the cell.
A. M Yu, J. B. Lucks*. Nature Strucutral and Molecular Biology. (2019). doi: 10.1038/s41594-019-0213-2.
Links: Journal, PDF, SharedIT (Free)

Our news-and-views piece for Sun et al.’s report “RNA structure maps across mammalian cellular compartments” in Nature Chemical Biology. We highlight what we think is the most interesting finding, that RNA folds established near chromatin can exist throughout the cellular lifecycle of the RNA, potentially pointing to the importance of cotranscriptional folding for determining functional RNA states.

51. PLANT-Dx: A molecular diagnostic for point of use detection of plant pathogens.
M. Verosloff, J. Chappell, K. L. Perry, J. R. Thompson, J. B. Lucks*. ACS Synthetic Biology. (2019). doi: 10.1021/acssynbio.8b00526 
Links: Journal, PDF, Supplemental Info, BioRxiv (open access), Blog Post

In this work, we demonstrate the potential of Point of use LAb iN a Tube (PLANT-dx). This system enables robust and user-friendly detection of plant pathogens by tying together isothermal amplification and modular RNA regulators within cell-free extracts. By running off of the end-user’s body heat, PLANT-dx takes input plant lysate and generates a colorimetric output in response to the presence of a target pathogen’s genomic material. We hope this work enables greater autonomy and decision making for farmers, especially those in rural and resource limited areas.

50. Elements of RNA Design.
P. D. Carlson, J. B. Lucks. Biochemistry. (2019). doi: 10.1021/acs.biochem.8b01129
Links: Journal, PDF, Blog Post

Our perspective on the RNA design-build-test-learn cycle, and what is needed to unlock even more powerful potential for designed RNAs.

49. Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry.
A. D. Silverman, N. Kelley-Loughnane, J. B. Lucks, M. C. Jewett. ACS Synthetic Biology. (2019). doi: 10.1021/acssynbio.8b00430
Links: Journal, PDF, Supplemental Info, BioRxiv (Open Access) Blog Post

This paper investigates the link between how a bacterial extract is made and its function, specifically finding that the addition of a few postlysis processing steps are necessary for activation of transcription in the final extract. The finding has great impact on the design of cell-free biosensors that rely on a transcriptional regulatory mechanism.

48. Snapshot: RNA Structure Probing Technologies.
P. D. Carlson, M. E. Evans, A. M Yu, E. J. Strobel, J. B. Lucks. Cell. (2018). doi: 10.1016/j.cell.2018.09.024
Links: Journal, PDF, Blog Post

This two-page SnapShot highlights the RNA chemical probing workflow, biological questions that can be informed by these experiments, and some options for probes.

47. High-throughput determination of RNA structures.
E. J. Strobel, A. M Yu, J. B. Lucks. Nature Reviews Genetics. (2018). doi: 10.1038/s41576-018-0034-x
Links: Journal, PDF, Blog Post

A comprehensive review of high throughput sequencing-based techniques to characterize RNA structures. This review includes a historical perspective, an overview of experimental approaches, discussion of data analysis approaches and highlights for applications to a range of questions in RNA biology. We also include a perspective on what is needed to make these approaches more accurate and more powerful.

46. Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.
A. Westbrook#, X. Tang#, R. Marhsall, C. Maxwell, J. Chappell, D. Agrawal, M. Dunlop, V. Noireaux, C. Beisel, J. B. Lucks*, E. M. Franco*. Biotechnology and Bioengineering. (2019). doi: 10.1002/bit.26918 # = Equal contribution
Links: Journal, PDF, BioRxiv

In this paper, we build an incoherent feed-forward loop—a network motif that produces a characteristic “pulse” in gene expression—out of RNA regulators by combining STAR transcriptional activation with CRISPRi transcriptional repression. We also demonstrate the importance of characterizing the dynamics of these regulators, using cell-free extracts as an easily manipulated modelling testbed.

45. A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation.
C. J. Glasscock, L. E. Yates, T. Jaroentomeechai, J. D. Wilson, J. H. Merritt, J. B. Lucks, M.P. DeLisa. Metabolic Engineering. (2018). doi: 10.1016/j.ymben.2018.04.014
Links: Journal, PDF, Blog Post

In this paper we report the development of a flow cytometry assay that can be used to detect the presence of glycosylated proteins on cell surfaces. We then use it to optimize an E. coli-based glycosylation pathway.

44. Mathematical modeling of RNA-based architectures for closed loop control of gene expression.
D. K. Agrawal, X. Tang, A. Westbrook, R. Marshall, C. Maxwell, J. B. Lucks, V. Noireaux, C. L. Beisel, M. Dunlop, E. Franco. ACS Synthetic Biology. (2018). doi: 10.1021/acssynbio.8b00040
Links: Journal, PDF, Blog Post

This collaborative computational study presents two designs for achieving biological control—for instance, to track the setpoint of the concentration of a given species and reject disturbances—by using RNA regulators to turn ON and OFF feedback-regulated genes.

43. Engineering a functional small RNA negative autoregulation network with model-guided design.
C. Hu, M. K. Takahashi, Y. Zhang , J. B. Lucks. ACS Synthetic Biology. (2018). doi: 10.1021/acssynbio.7b00440
Links: Journal, PDF, bioRxiv (Open Access), Blog Post

In this paper, we construct a negative auto-regulator—a network motif that stabilizes the response to input stimuli and reduces the time required to reach steady-state—using RNA regulators. The design is functional when prototyped in cell-free extracts and then ported into bacterial cells.

42. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.
K. E. Watters, K. Choudhary, S. Aviran, J. B. Lucks, K. L. Perry, J. R. Thompson. Nucleic Acids Research. (2017). doi: 10.1093/nar/gkx1273
Links: Jounral, PDF, SHAPE-Seq DataSets: RMDB Entries

Our report on the SHAPE-Seq-determined structure of the cucumber mosaic virus and how RNA structural elements are selected for for viral infectivity.

41. Computational design of Small Transcription Activating RNAs (STARs) for versatile and dynamic gene regulation.
J. Chappell, A. M. Westbrook, M. Verosloff, J. B. Lucks. Nature Communications. (2017). doi: 10.1038/s41467-017-01082-6
Links: Journal, PDF, BioRxiv (Open Access), Blog Post

Our STARS 2.0 paper highlights computational methods for designing libraries small transcription activating RNAs. We report STARs that demonstrate >1000-fold transcription activation and show that they are compatible with a variety of other circuits and mechanisms, including controlling metabolic flux and the synthesis of CRISPR guide RNAs.

40. Characterizing the structure-function relationship of a naturally- occurring RNA thermometer.
S. Meyer, J. B. Lucks. Biochemistry. (2017). doi: 10.1021/acs.biochem.7b01170
Links: Journal, PDF, BioRxiv (Open Access), SHAPE-Seq DataSets: RMDB Entries

Temperature-sensing RNAs known as RNA thermometers can regulate heat shock responses in bacteria. In this paper, we use SHAPE-Seq to observe how the agsA thermometer’s structure shifts in response to temperature to carry out this function.

39. Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.
E. J. Strobel, K. E. Watters, Y. Nedialkov, I. Artismovitch, J. B. Lucks. Nucleic Acids Research. (2017). doi: 10.1093/nar/gkx233
Links: Journal, PDF, BioRxiv (Open Access), SHAPE-seq DataSets: RMDB Entries, Sequencing Reads: SRA Entries, Blog Post

This paper describes a cotranscriptional SHAPE-seq method to reduce the labor required to generate transcripts of each intermediate length. Instead of cloning a transcription template with an EcoRIE111Q roadblock site for each length, this method uses templates with up to one biotin randomly located, which, when bound to streptavidin, blocks transcription.

38. Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator.
A. M. Westbrook, J. B. Lucks. Nucleic Acids Research. (2017). doi: 10.1093/nar/gkx215
Links: Journal, PDF, BioRxiv (Open Access), Blog Post

In this work, we combine the pT181 RNA repressor with a toehold switch to enable the design of a trans regulator that simultaneously blocks both transcription and translation. The final dual-controlled designed can achieve >95% specific repression of target genes when implemented in genetic circuits.

37. Turning it up to 11: Modular proteins amplify RNA sensors for sophisticated circuitry.
J. Chappell, J. B. Lucks. Cell Systems. (2016). doi: 10.1016/j.cels.2016.12.004
Links: Journal, PDF, Blog Post

Our perspective on Wang et al. “Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification” in Cell Systems. We describe how important it is to match transfer curves for correct genetic circuit function.

36. Cotranscriptional folding of a riboswitch at nucleotide resolution.
K. E. Watters#, E. J. Strobel#, A. M. Yu, J. T. Lis, J. B. Lucks. Nature Structure and Molecular Biology. (2016). doi: 10.1038/nsmb.3316 # = Equal contribution
Links: Journal, PDF, SHAPE-Seq DataSets: RMDB Entries, Sequencing Reads: SRA Entries, Blog Posts: Our cotranscriptional SHAPE-Seq paper is recommended by F1000 Prime! and Our paper on cotranscriptional SHAPE-Seq is published!

This work demonstrates a novel approach of combining our previous SHAPE-seq RNA structure probing method with in vitro transcriptional roadblocks to observe how RNA folds during transcription. Using this method, we observed intermediate structural states of the signal recognition particle RNA and the fluoride riboswitch.

35. Mapping RNA structure in vitro with SHAPE chemistry and next-generation sequencing (SHAPE-Seq).
K. E. Watters, J. B. Lucks. in RNA Structure Determination: Methods and Protocols, D. H. Turner, D. H. Mathews (eds.), Methods in Molecular Biology, 2016, 1490, 135-162. doi: 10.1007/978-1-4939-6433-8_9
Links: Book, PDF

A book chapter on in vitro SHAPE-Seq protocols.

34. Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.
M. K. Takahashi, K. E. Watters, P. M. Gaspar, T. R. Abbott, P. D. Carlson, A. A. Chen, J. B. Lucks. RNA. (2016). doi: 10.1261/rna.054916.115
Links: Journal, PDF, SHAPE-Seq DataSets: RMDB Entries, Blog Post

Here we use SHAPE-Seq to interrogate a series of engineered RNA transcriptional attentuators to find reactivity signatures that correlate with function. We then use these principles to design new attenuators that can function in cells. Molecular dynamics simulations are used to reveal a critical need for hairpin stem flexibility for transcriptional attenuator function.

33. RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs.
E. J. Strobel, K. E. Watters, D. Loughrey, J. B. Lucks. Current Opinion in Biotechnology. (2016). doi: 10.1016/j.copbio.2016.03.019
Links: Journal, PDF, Blog Post

A review of current discoveries in RNA biology, including an overview of high throughput experimental methods for uncovering RNA structures.

32. Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).
K. E. Watters, A. M. Yu, E. J. Strobel, A. H. Settle, J. B. Lucks. Methods. (2016). doi: 10.1016/j.ymeth.2016.04.002
Links: Journal, PDF, BioRxiv (Open Access), SHAPE-seq DataSets: RMDB Entries, Blog Post

This methods paper provides the details to understand and perform SHAPE-seq v2.1 in cells and in vitro. Two key improvement over v2.0 are increased flexibility in barcoding the cDNA library for sequencing and a selective PCR step in library preparation to reduce unwanted side products.

31. Engineered Protein Machines: Emergent Tools for Synthetic Biology.
C. J. Glasscock, J. B. Lucks, M. P. DeLisa. Cell Chemical Biology. (2016). doi: 10.1016/j.chembiol.2015.12.004
Links: Journal, PDF, Blog Post

This review article highlights the progress made in understanding, engineering and repurposing bacterial protein machines for use in synthetic biology. In particular, it discusses efforts in engineering DNA/RNA replication and synthesis machinery, orthogonal ribosomes, as well as protein folding and translocation machinery. The article ends with applications of these synthetic machineries in biotechnology such as the development of new therapeutics and vaccines.

30. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.
K. E. Watters, Timothy R. Abbott, J. B. Lucks. Nucleic Acids Research. (2015). doi: 10.1093/nar/gkv879
Links: Journal, PDF, SHAPE-Seq DataSets: RMDB Entries, Blog Post

In this paper, a method is developed to characterize RNA structure and function simultaneously with SHAPE-seq and functional assays. This approach is then applied to correlate RNA structure with function of synthetic RNA-RNA regulatory pairs and endogenous RNAs in E. coli.

29. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.
S. Meyer, J. Chappell, S. Sankar, R. Chew, J. B. Lucks. Biotechnology and Bioengineering. (2015). doi: 10.1002/bit.25693
Links: Journal, PDF, bioRxiv (Open Access), Blog Post: Sarai’s paper featured in B&B spotlight! and Improving STARs with rational RNA engineering strategies.

Here, we demonstrate varies strategies for improving upon the capacity of everyone’s favorite RNA regulators, small transcription activating RNAs (STARs). We showcase general rules for enhancing STARs’ fold activity through the combination of expression level tuning and RNA engineering design. These enhanced antisense regulators continue to demonstrate their tremendous orthogonality against both each other and panels of antisense RNA repressors. This work serves to augment the utility of the STARs toolkit providing researchers with a means for precision control over transcription-level gene expression.

28. A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future.
J. Chappell, K. E. Watters, M. K. Takahashi, J. B. Lucks. Current Opinion in Chemical Biology. (2015). doi: 10.1016/j.cbpa.2015.05.018
Links: Journal, PDF, Blog Post

Advances in RNA mechanism design tools have expanded the dynamic range and orthogonality of RNA-based synthetic mechanisms. This paper explains how these improvements can expand RNA genetic circuit applications, including those in metabolic engineering and diagnostics. It then describes how experimental and computational RNA structural tools can further improve our understanding of these mechanisms.

27. Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.
M. K. Takahashi#, C. A. Hayes#, J. Chappell, Z. Z. Sun, R. M. Murray, V. Nouireaux*, J. B. Lucks*. Methods. (2015).  doi: 10.1016/j.ymeth.2015.05.020 # = Equal contribution
Links: Jounral, PDF, BioRxiv (Open Access)

This paper, born out of a Cold Spring Harbor laboratory course, describes how to use cell-free transcription-translation systems to rapidly build and test RNA-regulated genetic circuits, including descriptions of simple experiments, important controls, and how to interpret the data.

26. Generating effective models and parameters for RNA genetic circuits.
C. Y. Hu, J. D. Varner, J. B. Lucks. ACS Synthetic Biology. (2015). doi: 10.1021/acssynbio.5b00077
Links: Journal, PDF, BioRxiv (Open Access), Code: GitHub, Blog Post

This paper seeks to understand how to parameterize models of RNA regulation using E. coli cell-free extracts by outlining a defined set of simple experiments that can rapidly and quantitatively inform new designs. These models can effectively predict the performance of a transcriptional genetic cascade, when provided the network topology and the concentration of each component.

25. Creating Small Transcription Activating RNAs.
J. Chappell, M. K. Takahashi, J. B. Lucks. Nature Chemical Biology. (2015). doi: 10.1038/NCHEMBIO.1737
Links: Journal, PDF, Supplemental Info, Blog Posts: STARs are born in Nature Chemical Biology!, STARs are on the front page of the Nature Chemical Biology Website!, STARs Paper Among Most Read on Nature Chemical Biology Site!
See Commentary! N. Rusk. “Synthetic biology: RNA that activates transcription.”, Nature Methods, 2015, 12, 290.

This is the first work to create and characterize small transcription activating RNAs, known as STARS. STARS are antisense RNAs that activate transcription of their target by disrupting a terminator in the sense RNA. This work demonstrates the versatility of STARs in cells and in vitro, as well as on both natural and synthetic target RNAs.

24. SHAPE-Seq 2.0: Systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next-generation sequencing.
D. Loughrey#, K. E. Watters#, A. Settle, J. B. Lucks. Nucleic Acids Research. (2014). (* Co-first author) doi: 10.1093/nar/gku909 # = Equal contribution
Links: Journal, PDF, SHAPE-Seq DataSets: RMDB Entries.

In this paper, we analyze and optimize SHAPE-seq, a technique for probing RNA structures. The results present an updated SHAPE-Seq protocol, v2.0, which can obtain reactivity information for every nucleotide of an RNA without requiring an internal RT priming site.

23. Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems.
M. K. Takahashi, J. Chappell, C. A. Hayes, Z. Z. Sun, J. Kim, V. Singhal, K. J. Spring, S. Al-Khabouri, C. P. Fall, V. Noireaux, R. M. Murray, J. B. Lucks. ACS Synthetic Biology. (2014). doi: 10.1021/sb400206c
Links: Journal, PDF, BioRxiv (Open Access)

In this paper, an E. coli-based cell free transcription-translation (TX-TL) system is adapted for prototyping RNA genetic networks. This work demonstrates that the response time for an RNA cascade is approximately 5 minute, which allows for more rapid prototyping than protein-based circuits. By prototyping a single input molecule (SIM) RNA circuit in this system, the first in vivo SIM RNA circuit is created and characterized.

22. The Centrality of RNA for Engineering Gene Expression.
J. Chappell, M. K. Takahashi, S. Meyer, D. Loughrey, K. Watters, J. B. Lucks. Biotechnology Journal. (2013). doi: 10.1002/biot.201300018
Links: Journal, PDF

This review focuses on the role of RNA in synthetic biology and highlights novel methodology for interrogating the form and function relationship of this amazing oligonucleotide. We highlight the numerous RNA regulators of gene expression and delve into methods for engineering synthetic variants. This leads to discussions on combining the power of next-gen sequencing with chemical probing in order to gain insights on RNA structure and how it translates into overall RNA function. This and similar technologies can also be used within the context of the cell to answer fundamental biological questions surrounding cellular RNA.

21. A modular strategy for engineering orthogonal chimeric RNA transcription regulators.
M. K. Takahashi, J. B. Lucks. Nucleic Acids Research. (2013). doi: 10.1093/nar/gkt452
Links: Journal, PDF

This work develops a set of orthogonal sense-antisense RNA pairs by engineering chimeric attenuators from naturally-occurring translation regulators, expanding the toolkit for RNA genetic circuitry engineering.

20. SHAPE-Seq: High Throughput RNA Structural Analysis.
S. A. Mortimer, C. Trapnell, S. Aviran, L. Pachter, J. B. Lucks. Current Protocols in Chemical Biology. (2012). doi: 10.1002/9780470559277.ch120019
Links: Journal, PDF

The first detailed protocol of SHAPE-Seq.

19. An adaptor from translational to transcriptional control enables predictable assembly of complex regulation.
C. C. Liu, L. Qi, J. B. Lucks, T. H. Segall-Shapiro, D. Wang, V. Mutalik, A. P. Arkin. Nature Methods. (2012). doi: 10.1038/nmeth.2184
Links: Journal, PDF
See Commentary! J. J. Tabor. “Modular gene-circuit design takes two steps forward.” Nature Methods, 2012, 9, 1061-1063.

By configuring the RNA-In/Out system to regulate the translation of a leader-peptide attenuation system, this work demonstrated a new strategy for combining the orthogonality of RNA translational regulation with mechanisms to allow the control of transcription.

18. The Stanford RNA Mapping Database for sharing and visualizing RNA structure mapping experiments.
P. Cordero, J. B. Lucks, R. Das. Bioinformatics. (2012). doi: 10.1093/bioinformatics/bts554
Links: Journal, PDF, Arxiv (q-bio.BM)

This paper launched the RMDB – a database for high throughput chemical probing data that is a growing resource for both experimental and computational biologists interested in RNA structure. The RMDB can be accessed at https://rmdb.stanford.edu/

17. Rationally designed families of orthogonal RNA regulators of translation.
V. K. Mutalik, L. Qi, J. C. Guimaraes, J. B. Lucks, A. P. Arkin. Nature Chemical Biology. (2012). doi: 10.1038/nchembio.919
Links: Journal, PDF

This paper was amongst the first to systematically solve the orthogonality problem – how do you design regulatory molecules to regulate specific targets and not cross-talk with each other – for RNAs. By focusing on the RNA-In/Out translation repression systems, we found sequence determinants in specific regions of the regulatory RNAs that when mutated could be used to create the largest at the time mutually orthogonal set of regulators.

16. Engineering naturally occurring trans-acting non-coding RNAs to sense cellular signals.
L. Qi, J. B. Lucks, C. C. Liu, V. K. Mutalik, A. P. Arkin. Nucleic Acids Research. (2012). doi: 10.1093/nar/gks168
Links: Journal, PDF

This paper presents a strategy to fuse protein/ligand-binding RNA aptamers to non-coding RNAs to make protein/ligand-responsive non-coding RNA switches. This strategy works with non-coding RNAs that control transcription and translation and can be used to incorporate protein/ligand triggers for RNA-only circuits.

15. RNA Structure Characterization from Chemical Mapping Experiments.
S. Aviran, J. B. Lucks, L. Pachter. Forty-Ninth Allerton Conference, UIUC Illinois, 1743-1750.(2011). Conference PDF DOI: 10.1109/Allerton.2011.6120379 (FREE)
Links: Conference PDF, Arxiv(q-bio)

An updated maximum likelihood model for SHAPE-Seq reactivities from Aviran et. al, PNAS, 2011. This much simpler derivation is easier to extend to different experimental scenarios (such as multiple reverse transcriptase primers) and introduces a new quantity, beta-reactivities, which are easier to interpret.

14. Multiplexed RNA structure characterization with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).
J. B. Lucks*, S. A. Mortimer, C. Trapnell, S. Luo, S. Aviran, G. P. Schroth, L. Pachter, J. A. Doudna*, A. P. Arkin*. PNAS. (2011). (* Co-corresponding author) doi: 10.1073/pnas.1106501108
Links: Journal, PDF
See Commentary! K. M. Weeks. “RNA Structure Probing dash Seq.”, PNAS, 108, 10933, 2011.

This paper presents the first method to capture nucleotide-resolution structural information for hundreds of RNAs in a single experiment. This method, called SHAPE-Seq, works by merging chemical probing of RNA structures, with next-generation sequencing to read out modification patterns and infer structures. It opened the door to very high throughput RNA structural biology and it and similar techniques are used by laboratories around the world to address fundamental questions in RNA biology and RNA engineering.

13. Modeling and automation of sequencing-based characterization of RNA structure.
S. Aviran, C. Trapnell, J. B. Lucks, S. A. Mortimer, S. Luo, G. P. Schroth, J. A. Doudna, A. P. Arkin, L. Pachter. PNAS. (2011). doi: 10.1073/pnas.1106541108
Links: Journal, PDF
See Commentary! K. M. Weeks. “RNA Structure Probing dash Seq.”, PNAS, 108, 10933, 2011.

This work describes a new approach for analyzing next generation sequencing datasets from high throughput RNA structure chemical probing experiments. Importantly it was the first approach that allowed fully automated analysis, and presents a maximum-likelihood framework for estimating this structural information that has been extended in a number of important works.

12. Versatile RNA-sensing transcriptional regulators for engineering genetic networks.
J. B. Lucks, L. S. Qi, V. K. Mutalik, D. Wang, A. P. Arkin. PNAS. (2011). doi: 10.1073/pnas.1015741108
Links: Journal, PDF

This paper describes an innovation in RNA synthetic biology. By engineering non-coding RNA regulators that control transcription, we built the first ‘RNA-only’ genetic networks. This opens the door for cellular information processing through RNA circuits.

11. Synthetic Biology’s Hunt for the Biological Transistor.
J. B. Lucks, A. P. Arkin. IEEE Spectrum, March, 38, 2011 (Coverstory).
Links: PDF

How is a programming a biological system like programming a computer? Find out from this introduction to synthetic biology piece.

10. Why on Earth?: Evaluating Hypotheses About the Physiological Functions of Human Geophagy.
S. L. Young, P. W. Sherman, J. B. Lucks, G. H. Pelto. Quarterly Review of Biology. (2011). PMID: 21800636.
Links: Journal, PDF

A systematic literature review of reports of pica – the purposeful consumption of non-food items – is used to evaluate three key hypotheses about what causes this behavior in humans.

9. Evolution, ecology and the engineered organism: lessons for synthetic biology.
Jeffery M. Skerker, J. B. Lucks, A. P. Arkin. Genome Biology.(2009). doi: 10.1186/gb-2009-10-11-114
Links: Journal, PDF

As the scope and complexity of synthetic biology grows, an understanding of evolution and ecology will be critical to its success.

8. Toward scalable parts families for predictable design of biological circuits.
J. B. Lucks, L. S. Qi, W. Whitaker, A. P. Arkin. Current Opinion in Microbiology. (2008). doi: 10.1016/j.mib.2008.10.002
Links: Journal

A core concept in synthetic biology is decomposing biological function into elementary ‘parts’ that can composed together to create higher order function. This review outlines the properties of parts necessary to complete this vision: independence, reliability, tunability, orthogonality, and composability.

7. Python – All a Scientist Needs.
J. B. Lucks (2008).
Links: OOW: Article Page, Arxiv (q-bio.QM), Video: Pycon 2008 Presentation

The python programming environment offers many useful tools for scientists.

6. Genome landscapes and bacteriophage codon usage.
J. B. Lucks, D. R. Nelson, G. Kudla, J. B. Plotkin. PLoS Computational Biology. (2008). doi: 10.1371/journal.pcbi.1000001
Links: Journal, PDF, Arxiv(q-bio.GN)

This paper uses the concept of a ‘genome landscape’ – the DNA unzipping energy landscape derived from phage genome sequences – to examine long range patterns in phage codon usage. We find some phages exhibit non-random patterns in GC content and codon usage related to where structural protein genes are located.

5. Dynamics of RNA Translocation through a Nanopore.
J. B. Lucks, Y. Kafri. (2007).
Links: PDF, Arxiv

This manuscript presents an energy landscape model of DNA unzipping as it applies to threading one strand of a DNA duplex through a nanopore. This early calculation was done to investigate DNA nanopore sequencing.

4. Crystallography on Curved Surfaces.
V. Vitelli, J. B. Lucks, D. R. Nelson. PNAS. (2006). doi: 10.1073/pnas.0602755103
Links: Journal, PDF, Arxiv

Here we report a framework for solving how topological defects interact with curvature in a 2D-crystalline system. Analytical solutions of the Foppl-von Karman equations lead to an effective ‘electrostatic’ theory for defect interactions. This work has implications for viral capsid structure.

3. Pause Point Spectra in DNA Constant-Force Unzipping.
J. D. Weeks, J. B. Lucks, Y. Kafri, C. Danilowicz, D. R. Nelson and M. Prentiss. Biophysical Journal. (2005). doi: 10.1529/biophysj.104.047340
Links: Journal, PDF, Arxiv

This paper describes a statistical mechanical calculation approach to examining single molecule DNA unzipping experiments. Monte carlo simulations on a DNA unzipping free-energy landscape were shown to obey d hopping dynamics similar to experimental observations.

2. Constructing a map from the electron density to the exchange-correlation potential.
J. B. Lucks, A. J. Cohen, N. C. Handy. Physical Chemistry Chemical Physics. (2002).
Links: Journal, PDF

Here we report a new approach to numerically interpolating exchange-correlation potentials calculated from the Zhao-Morrison-Parr method.

1. Constructing exact density functionals from the moments of the electron density.
P. W. Ayers, J. B. Lucks, R. G. Parr. Acta Univ. Debrecceniensis Series Physica et Chimica, XXXIV-XXXV, 223, (2002).
Links: PDF

Here we report a new technique to numerically compute kinetic energy density functionals using a moment expansion of the electron density .