Zhixia Liu

Verified email at gmail.com

The Scripps Research Institute




Protein folding, Protein engineering, Aptamer discovery, Drug delivery


Scopus Publications

Scopus Publications

  • Selection of Aptamers with Large Hydrophobic 2′-Substituents
    Qian Shao, Tingjian Chen, Kai Sheng, Zhixia Liu, Zhuochen Zhang, and Floyd E. Romesberg

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 142, Pages: 2125-2128, Published: 5 February 2020 American Chemical Society (ACS)
    Previously, we evolved a DNA polymerase, SFM4-3, for the recognition of substrates modified at their 2' positions with a fluoro, O-methyl, or azido substituent. Here we use SFM4-3 to synthesize 2'-azido-modified DNA; we then use the azido group to attach different, large hydrophobic groups via click chemistry. We show that SFM4-3 recognizes the modified templates under standard conditions, producing natural DNA and thereby allowing amplification. To demonstrate the utility of this remarkable property, we use SFM4-3 to select aptamers with large hydrophobic 2' substituents that bind human neutrophil elastase or the blood coagulation protein factor IXa. The results indicate that SFM4-3 should facilitate the discovery of aptamers that adopt novel and perhaps more protein-like folds with hydrophobic cores that in turn allow them to access novel activities.

  • Selection of 2′-Fluoro-Modified Aptamers with Optimized Properties
    Deepak Thirunavukarasu, Tingjian Chen, Zhixia Liu, Narupat Hongdilokkul, and Floyd E. Romesberg

    Journal of the American Chemical Society, ISSN: 00027863, eISSN: 15205126, Volume: 139, Pages: 2892-2895, Published: 1 March 2017 American Chemical Society (ACS)
    RNA or single-stranded DNA aptamers with 2'-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2'-modifications, we now report the selection of 2'-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity. Interestingly, the 2'-F substituents facilitate the selection of specific interactions with HNE and overcome nonspecific electrostatic interactions that can otherwise dominate. The data demonstrate that inclusion of only a few 2'-F substituents can optimize properties far beyond simple nuclease resistance and that SFM4-3 should prove valuable for the further exploration and production of aptamers with properties optimized for various applications.

  • Evolved polymerases facilitate selection of fully 2′-OMe-modified aptamers
    Zhixia Liu, Tingjian Chen, and Floyd E. Romesberg

    Chemical Science, ISSN: 20416520, eISSN: 20416539, Pages: 8179-8182, Published: 2017 Royal Society of Chemistry (RSC)
    Evolved DNA polymerases are used in selections with fully 2′-OMe modified libraries to identify aptamers with high affinity for HNE.

  • The expanding world of DNA and RNA
    Tingjian Chen, Narupat Hongdilokkul, Zhixia Liu, Deepak Thirunavukarasu, and Floyd E Romesberg

    Current Opinion in Chemical Biology, ISSN: 13675931, eISSN: 18790402, Pages: 80-87, Published: 1 October 2016 Elsevier BV
    DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.

  • Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA
    Tingjian Chen, Narupat Hongdilokkul, Zhixia Liu, Ramkrishna Adhikary, Shujian S. Tsuen, and Floyd E. Romesberg

    Nature Chemistry, ISSN: 17554330, eISSN: 17554349, Pages: 556-562, Published: 1 June 2016 Springer Science and Business Media LLC
    The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, the use of these aptamers is limited in many applications by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases but cannot be synthesized by natural polymerases. Here, we report the development of a polymerase evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe modified oligonucleotides and their DNA counterparts via “transcription” and “reverse transcription,” or more importantly, PCR amplify partially C2'-OMe or C2'-F modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides was evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for the tailoring of polymerases to have other types of novel function.

  • Capture and enumeration of mRNA transcripts from single cells using a microfluidic device
    Matthew T. Walsh, Alexander P. Hsiao, Ho Suk Lee, Zhixia Liu, and Xiaohua Huang

    Lab on a Chip, ISSN: 14730197, eISSN: 14730189, Pages: 2968-2980, Published: 21 July 2015 Royal Society of Chemistry (RSC)
    We report an integrated microfluidic device for direct capture and digital counting of polyadenylated mRNA molecules from single cells.

  • Strengthening the stability of a tunnel-shaped homotetramer protein with nanogels
    Zhixia Liu, Diannan Lu, Ling Yin, Jianmin Li, Yuanchen Cui, Wei Chen, and Zheng Liu

    Journal of Physical Chemistry B, ISSN: 15206106, eISSN: 15205207, Volume: 115, Pages: 8875-8882, Published: 21 July 2011 American Chemical Society (ACS)
    Urate oxidase (UOX, EC is effective for the treatment of gout and hyperuricaemia associated with tumor lysis syndrome. The inherent poor stability of UOX to temperature, proteolysis, and acidic environments is known to limit its efficacy. Herein, we encapsulated UOX into spherical and porous nanogels with diameters of 20-40 nm via a two-step in situ polymerization in the presence of oxonic acid potassium salt, an inhibitor of UOX. The UOX nanogel retained 70% of the initial activity but showed an expanded pH spectrum from pH 6-10 to 3-10 and an extended half-life at 37 °C from 5 min to 3 h. The enhanced pH stability, thermal stability, and enzyme resistance of the UOX nanogels were also confirmed by using fluorescence spectroscopy and enzymatic digestion. A molecular dynamics simulation was performed as a way to probe the mechanism underlying the formation of UOX nanogels as well as the strengthened stability against harsh conditions. It was shown that the encapsulation into the polyacrylamide network reinforced the intersubunit hydrogen bonding, shielded the hydrolytic reaction site, and thus protected the tertiary and quaternary structure of UOX. The UOX nanogel with enhanced stability provided a stable enzyme model that enables the exploration of UOX in the diagnosis and therapy of disorders associated with altered purine metabolism.

  • Recent advances in nanostructured biocatalysts
    Jun Ge, Diannan Lu, Zhixia Liu, and Zheng Liu

    Biochemical Engineering Journal, ISSN: 1369703X, Pages: 53-59, Published: 15 April 2009 Elsevier BV
    Abstract Recent years have witnessed a renaissance in the field of chemically re-engineering of enzymes to obtain highly selective and efficient biocatalysts for catalyzing processes under various conditions. The incorporation of enzyme into nanostructured materials is particularly noteworthy from a structural perspective since there are unprecedented opportunities in such systems to establish suitable microenvironments for chosen enzymes. This review summarizes recent developments in the nanostructured biocatalyst with emphasis on those formed with polymers. Based on the synthetic procedures employed, the established methods are grouped into three major categories—“grafting onto”, “grafting from”, and “self-assembly”. The merits of the methods in enhancing enzyme stability at adverse conditions and their potential for large-scale preparation and the use of the nanostructured biocatalysts are discussed. The molecular fundamentals underlying each method are highlighted, and the use of molecular simulation as a tool for the design and application of nanostructured biocatalysts, although at a nascent stage, is presented. Finally, the problems encountered with nanostructured biocatalysts are discussed together with the future prospects of such systems.

  • Strengthening intersubunit hydrogen bonds for enhanced stability of recombinant urate oxidase from Aspergillus flavus: Molecular simulations and experimental validation
    Zhixia Liu, Diannan Lu, Jianmin Li, Wei Chen, and Zheng Liu

    Physical Chemistry Chemical Physics, ISSN: 14639076, Pages: 333-340, Published: 2009 Royal Society of Chemistry (RSC)
    The aim of this study was to obtain molecular insight into the deactivation of recombinant urate oxidase (uricase, UOX, EC (rUOX) from Aspergillus flavus. The enzyme is a tunnel-shaped homotetramer and has important clinical applications. By means of molecular dynamics simulations, multidimensional structural characterization and enzyme activity assays, we concluded that the thermal deactivation of UOX at neutral pH was associated with the loss of intersubunit hydrogen (H) bonds. This mechanism could also explain the deactivation of dilute aqueous UOX. Thermal deactivation of aqueous UOX due to dissociation of its subunits was ruled out. Displacement of H(2)O from the surface of UOX by less polar solvents such as methanol and dimethyl sulfoxide (DMSO) was proposed as an approach for strengthening intersubunit H bonds and consequently UOX stability. The effectiveness of this method was validated by both in silico and in vitro experiments. The results mentioned above provide insights for improving the stability of UOX and extending its applications. They may also be helpful for understanding the properties of other multimeric proteins.

  • Role of intersubunit hydrogen bond to urate oxidase stability: Molecular simulation and experimental validation
    AIChE Annual Meeting, Conference Proceedings, Published: 2008

  • Robust biocatalytic single enzyme nanogels by versatile strategies
    2007 AIChE Annual Meeting, Published: 2007

  • Fabrication of single carbonic anhydrase nanogel against denaturation and aggregation at high temperature
    Ming Yan, Zhixia Liu, Diannan Lu, and Zheng Liu

    Biomacromolecules, ISSN: 15257797, Pages: 560-565, Published: February 2007 American Chemical Society (ACS)
    A two-step procedure to encapsulate a single bovine carbonic anhydrase (BCA) molecule into a spherical nanogel was proposed. BCA was reacted first with N-acryloxysuccinimide to introduce surface vinyl groups, followed by in-situ aqueous polymerization. Characterization of the nanogel by dynamic light scattering, transmission electron microscopy, and atomic force microscopy confirmed that each nanogel contained a single BCA molecule. The encapsulated BCA maintained 70% of the activity of its free counterpart, but exhibited an increase in the molten temperature from 64 to 81 degrees C demonstrated by differential scanning calorimetry and an extension of the half-life from less than 3 to over 90 min at 75 degrees C. Circular dichroism spectroscopy indicated that the encapsulation and the multi-point covalent linkage between BCA and the polymer shell strengthened the secondary structure and thus inhibited the aggregation at high temperature. The uniform BCA nanogel with enhanced structural stability against denaturation and aggregation expands the applications of BCA catalysis, particularly those carried out at high temperatures.

  • Dextran-grafted-PNIPAAm as an artificial chaperone for protein refolding
    Diannan Lu, Zhixia Liu, Minlian Zhang, Xiaogong Wang, and Zheng Liu

    Biochemical Engineering Journal, ISSN: 1369703X, Pages: 336-343, Published: January 2006 Elsevier BV
    A temperature stimuli-responsive polymer, dextran-grafted-PNIPAAm (DGP), was prepared by radical polymerization using cerium nitrate as the initiator. The structure and the grafting ratio of DGP were determined by FT-IR and elemental analysis. The temperature-stimuli responsive behavior of DGP and the size of the DGP self-assemblies at different temperatures were determined by transmittance and dynamic light scattering, respectively. The use of DGP as an artificial chaperone to assist protein refolding in vitro was demonstrated using hen egg white lysozyme (lysozyme) and carbonic anhydrase bovine (CAB) as model proteins. It was shown that the hydrophobic interaction between DGP and the protein being refolded gave a significant reduction in the rate of protein aggregation and a slight reduction in the refolding rate, and consequently gave an improved refolding yield. Moreover, the refolding of lysozyme and CAB at a temperature gradient starting above the LCST of DGP and ending at a lower temperature, which make DGP change from hydrophobic to hydrophilic, gave a significant increase in the refolding yield compared to that carried out at a constant temperature. The process mechanism of DGP assisted protein refolding was presented and the importance of controlling the hydrophobicity of the solution environment for protein refolding was discussed.

  • Molecular simulation of surfactant-assisted protein refolding
    Diannan Lu, Zheng Liu, Zhixia Liu, Minlian Zhang, and Pingkai Ouyang

    Journal of Chemical Physics, ISSN: 00219606, Volume: 122, Published: 2005 AIP Publishing
    Protein refolding to its native state in vitro is a challenging problem in biotechnology, i.e., in the biomedical, pharmaceutical, and food industry. Protein aggregation and misfolding usually inhibit the recovery of proteins with their native states. These problems can be partially solved by adding a surfactant into a suitable solution environment. However, the process of this surfactant-assisted protein refolding is not well understood. In this paper, we wish to report on the first-ever simulations of surfactant-assisted protein refolding. For these studies, we defined a simple model for the protein and the surfactant and investigated how a surfactant affected the folding behavior of a two-dimensional lattice protein molecule. The model protein and model surfactant were chosen such that we could capture the important features of the folding process and the interaction between the protein and the surfactant, namely, the hydrophobic interaction. It was shown that, in the absence of surfactants, a protein in an "energy trap" conformation, i.e., a local energy minima, could not fold into the native form, which was characterized by a global energy minimum. The addition of surfactants created folding pathways via the formation of protein-surfactant complexes and thus enabled the conformations that fell into energy trap states to escape from these traps and to form the native proteins. The simulation results also showed that it was necessary to match the hydrophobicity of surfactant to the concentration of denaturant, which was added to control the folding or unfolding of a protein. The surfactants with different hydrophobicity had their own concentration range on assisting protein refolding. All of these simulations agreed well with experimental results reported elsewhere, indicating both the validity of the simulations presented here and the potential application of the simulations for the design of a surfactant on assisting protein refolding.

  • Surfactant assisted protein refolding in vitro: Molecular simulation
    Huagong Xuebao/Journal of Chemical Industry and Engineering (China), ISSN: 04381157, Pages: 1063-1069, Published: June 2005

  • The mechanism of PNIPAAm-assisted refolding of lysozyme denatured by urea
    Diannan Lu, Zhixia Liu, Minlian Zhang, Zheng Liu, and Haimeng Zhou

    Biochemical Engineering Journal, ISSN: 1369703X, Pages: 55-64, Published: 15 May 2005 Elsevier BV
    Abstract The molecular interaction of a temperature stimuli-responsive polymer, poly-N-isopropyl acrylamide (PNIPAAm), with lysozyme of different status was studied with an emphasis on the application of PNIPAAm for protein refolding. The refolding of lysozyme was performed by directly diluting denatured lysozyme into a refolding buffer containing PNIPAAm, in which PNIPAAm with the weight average molecular weight of 22,000, denoted as M-PNI, gave the best refolding yield in terms of the recovery of lysozyme activity. The interaction between M-PNI and lysozyme was investigated using non-reductive SDS–PAGE, circular dichroism (CD), fluorescence emission spectroscopy, and reverse phase HPLC. It was shown that the use of M-PNI increased the secondary structures of lysozyme and reduced the formation of protein aggregate. The correctly folded lysozyme has a weaker hydrophobicity compared to the denatured lysozyme. The PNIPAAm-lysozyme complex dissociates once lysozyme is correctly folded. The increase in the operational temperature leads to increases in both the refolding yield and the apparent rate of refolding. Based on above experimental results, a kinetic model of the refolding, both with and without PNIPAAm, was determined and a molecular view of lysozyme refolding using PNIPAAm was presented.



    Liu Z, Chen T, Romesberg FE, Evolved polymerases facilitate selection of fully 2’-OMe-modified aptamers, Chemical Science, 2017, 8: 8179
    Thirunavukarasu D, Chen T, Liu Z, Hongdilokkul N, Romesberg FE, Selection of 2’-Fluoro-Modified Aptamers with Optimized Properties, Journal of the American Chemical Society, 2017, 139: 2892
    Chen T, Hongdilokkul N, Liu Z, Thirunavukarasu D, Romesberg FE, The expanding world of DNA and RNA, Current Opinion in Chemical Biology, 2016, 34: 80
    Chen T, Hongdilokkul N, Liu Z, Adhikary R, Tsuen SS, Romesberg FE, Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA, Nature Chemistry, 2016, 8: 556
    Walsh M, Hsiao A, Lee HS, Liu Z, Huang X, Capture and enumeration of mRNA transcripts from single cells using a microfluidic device, Lab on a Chip, 2015, 15: 2968
    Liu Z, Lu D, Yin L, Li J, Cui Y, Chen W, Liu Z, Strengthening the stability of a tunnel-shaped homotetramer protein with nanogels, The Journal of Physical Chemistry B, 2011, 115: 8875
    Liu Z, Lu D, Li J, Chen W, Liu Z, Strengthening intersubunit hydrogen bonds for enhanced stability of recombinant urate oxidase from Aspergillus flavus: Molecular simulations and experimental validation, Physical Chemistry Chemical Physics, 2009, 11: 333
    Ge J, Lu D, Liu Z, Liu Z, Recent advances in nanostructured biocatalysts, Biochemical Engineering Journal, 2009, 44: 53
    Yan M, Liu Z, Lu D, Liu Z, Fabrication of single carbonic anhydrase nanogel against denaturation and aggregation at high temperature, Biomacromolecules, 2007, 8: 560
    Lu D, Liu Z, Zhang M, Wang X, Liu Z, Dextran-grafted-PNIPAAm as an artificial chaperone for protein refolding, Biochemical Engineering Journal, 2006, 27: 336
    Lu D, Liu Z, Zhang M, Liu Z, Zhou H, The mechanism of PNIPAAm-assisted refolding of lysozyme denatured by urea, Biochemical Engineering Journal, 2005, 24: 55
    Lu D, Liu Z, Liu Z, Zhang M, Ouyang P, Molecular simulation of surfactant-assisted protein refolding, The Journal of Chemical Physics, 2005, 122: Art No.134902(1-11)
    Lu D, Wang J, Liu Z, Zhang M, Liu Z, Surfactant assisted protein refolding in vitro: molecular simulation, Journal of Chemical Industry and Engineering-China, 2005, 56: 1063
    Ge J, Yan M, Lu D, Liu Z, Liu Z, Preparation and Characterization of Single-Enzyme Nanogels, In: Wang P. (eds) Nanoscale Biocatalysis. Methods in Molecular Biology (Methods and Protocols), vol 743. Humana Press, 2011


    Yan M, Liu Z, Ge J, Liu Z, Zhang M, Ouyang P, Method for manufacturing nanoscale macromolecular biocatalytic particles containing horseradish peroxidase, Patent No. CN1904042; CN 100469873