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Enhancing multi-step DNA processing by solid-phase enzyme catalysis on polyethylene glycol coated beads Shaohua Li, Aihua Zhang, Kelly Zatopek, Saba Parvez, Andrew Fenn Gardner, Ivan Correa, Christopher J. Noren, and Ming-Qun Xu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00299 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018
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Bioconjugate Chemistry
Enhancing Multi-step DNA Processing by Solid-phase Enzyme Catalysis on Polyethylene Glycol Coated Beads Shaohua Li†, Aihua Zhang†, Kelly Zatopek†, Saba Parvez†, Andrew F. Gardner†, Ivan R. Corrêa Jr.†, Christopher J. Noren†, Ming-Qun Xu*† † New England Biolabs Inc., 240 County Road, Ipswich, Massachusetts 01938, USA *
[email protected] ABSTRACT: Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activities compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in the immobilized enzyme activity loss. In this work, we demonstrate through kinetic studies that flexible polyethylene glycol (PEG) moieties modifying the surface of magnetic beads improve the activity of covalently-immobilized DNA replication enzymes. PEG-modified immobilized enzymes were utilized in library construction for Illumina next-generation sequencing (NGS) increasing the read coverage across AT-rich regions.
INTRODUCTION DNA processing and modifying enzymes play essential roles in DNA repair and replication in both eukaryotic and prokaryotic cells,1-4 including DNA chain-end excision,5-7 extension,8-9 mismatch repair,2, 10 and end-joining ligation.11 DNA enzymes are widely used in biotechnological applications such as gene editing,12-14 gene therapy,15-16 and sequencing.17-19 Although most of these enzymes are utilized and stored in aqueous solutions, there has been a growing interest toward the covalent conjugation of DNA processing enzymes on the surface of solid supports, especially micrometer-sized particles.20-22 Micrometer-sized particles/beads have been widely utilized in biochemical research, for example, in nucleic acid or protein chromatographic separation and fluorescence detection.23-27 Immobilizing DNA enzymes on microbeads combines their DNA-processing catalytic activities with the unique properties of solid supports (e.g., high surface area, mechanical stability, superparamagnetism, conductivity, etc.), making them useful tools for biochemical and biomedical applications.22 The immobilization process can also improve enzyme long-term stability and allow easier enzyme removal after catalysis. One of the main challenge for immobilized (IM) DNA modifying enzymes is to achieve high levels of specific activity. To date, just a handful of enzymes, most of them acting on small molecule substrates, have been immobilized on nanoparticles and demonstrated improved activity.28-32 Very few studies have been carried out with DNA modifying enzymes on micrometer-sized particles. Compared of their soluble counterparts, IM DNA enzymes on microbeads generally lose enzymatic activity.33 The intrinsic hydrophobic nature of most support surfaces could result in enzyme conformational change and spatial confinement, which ultimately leads to partial enzyme deactivation. The morphology of the surface
may reduce substrate access to the enzyme active site, resulting in slower diffusion rates for large polymeric substrates,34 including nucleic acids. Understanding the causes for reduced enzymatic activities as well as improving conjugation and bead chemistries can have a major impact in the field of solidphase biocatalysts. In this research, we covalently immobilize DNA replication and modifying enzymes on magnetic microbeads using the SNAP-tag® technology. This strategy utilizes a genetic fusion of the protein of interest to the SNAP-tag protein, which is covalently conjugated to a surface modified with SNAP-tag reactive O6-benzylguanine (BG) functional groups. The conjugation reaction is highly specific and with predictable directionally and stoichiometry.35-39 We explored the modification of solid supports using polyethylene glycol (PEG) which has been broadly studied for its high hydrophilicity, flexibility and biocompatibility.40-42 We utilized PEG both as surface coating and as a spacer between the BG reactive group and the bead surface, with the goal of improving the activity of immobilized DNA enzymes. Kinetic studies were performed on both soluble and immobilized enzymes to investigate causes of activity loss. We also demonstrated the use of PEG-modified immobilized enzymes in sequential reactions and in library preparation for next-generation sequencing (NGS) using an Illumina platform.
RESULTS AND DISCUSSION Preparation of immobilized enzymes SNAP-tagged DNA modifying enzymes were covalently conjugated to magnetic beads derivatized with BG as shown in Scheme 1. Briefly, 1 µm magnetic beads with –COOH coated surface were activated by reaction with 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) and N-
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hydroxysuccinimide (NHS), followed by reaction with BGNH2 to form a BG-coated surface. The amount of BG groups Scheme 1. Immobilization of SNAP-tagged enzymes on magnetic beads containing BG and PEG groups
conjugated to the surface was calculated by subtracting the BG content before and after the reaction, and was determined to be on average 0.18 µmol/mg beads. The remaining NHSester groups were quenched by reaction with diethanolamine to prevent any non-specific bead conjugation in the subsequent steps. The BG-coated beads were then conjugated with the SNAP-tagged T4 DNA polymerase (T4 pol), T4 polynucleotide kinase (T4 PNK) or Taq DNA polymerase (Taq pol). The enzyme to bead ratio was set to 0.188 nmol enzyme:1 mg bead, wherein the molar ratio of enzyme:BG was approximately 1:1000. These conditions ensure near complete enzyme conjugation (minimal enzyme activity was detected on the supernatant after the conjugation reaction), and more importantly minimize enzyme-enzyme interactions on the bead surface. The amount of enzyme immobilized in each case was quantified by a bicinchoninic acid (BCA) assay43 and the results are listed in Table S1. From the results, the majority of the SNAP-tagged enzymes (>90%) are immobilized on the beads, which demonstrates that the efficiency of SNAP-tag and BG conjugation is very high and are thus suitable for the immobilization application. To evaluate the exonuclease and polymerase activity of IM T4 pol on BG beads, we measured both the polynucleotide degradation and deoxynucleotide (dNTP) incorporation activity of SNAP-tagged T4 pol before and after conjugation. A mixture of two 5’ fluorescein (FAM) labeled double strand DNA (dsDNA) substrates (5’FAM-54mer/50mer and 5’FAM66mer/70mer) was used (Figure 1a, Table S2). An active T4 pol will add 4 nucleotides (nt) to the 3' end of the 66mer in the 5’FAM-66mer/70mer substrate (by means of its 3’-5’ poly-
merase activity) and excise the protruding 4 nt from the 3' end of the 54mer from the 5’FAM-54mer/50mer substrate (by means of its 3'-5' exonuclease activity) to produce the ds blunt end products 5’FAM-70 nt and 5’FAM-50 nt, respectively (Figure 1a). The polymerase to substrate molar ratio used was approximately 1:67. Reactions were stopped after 30 min incubation. Capillary electrophoresis (CE) was used to separate and quantify the fluorescently labeled substrates and products (Figure 1).44 As shown in Figure 1, soluble SNAP-tagged T4 pol (T4 pol_SNAP) almost completely converted both overhang substrates to blunt end products, demonstrating that the fusion to SNAP-tag does not have a detrimental effect on the T4 pol activity. However, unreacted substrates were still observed after 30 min reaction for T4 pol immobilized on BGcoated beads (Figure 1, IM T4 pol_BG-NH2). The conversion of each of the two FAM labeled substrates to the corresponding products was approximately 70%, indicating some degree of activity loss after T4 pol immobilization. Bead surface modification with hydrophilic PEG moieties We next sought to improve the specific activity of the immobilized enzymes by utilizing PEG during bead surface modification. As depicted in Scheme 1 (bottom approach), following bead functionalization with BG-NH2, a methoxypolyethylene glycol amine (Scheme 1, red sphere chain) was used to replace diethanolamine and react with any remaining active NHS-ester groups. Bead-coating with a flexible PEG chain is
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Bioconjugate Chemistry
expected to create a hydrophilic environment surrounding the IM enzymes. In addition, we hypothesize that the activity of
Figure 1. a) Representative CE traces for exonuclease and polymerase activities of soluble and immobilized T4 pol. The 54 nt and 66 nt peaks correspond to substrates (S), and the 50 nt and 70 nt peaks to products (P). b) DNA exonuclease and c) polymerase activities of soluble and immobilized T4 pol. IM T4 pol was prepared from beads functionalized with either BG-NH2 or with a BG-PEGn-NH2 with variable PEG length, and subsequently coated with either diethanolamine or with a methoxypolyethylene glycol amine with average molecular weight of 750 or 5000 Da (PEG 750 and PEG 5000, respectively). NCT, negative control in absence of enzyme.
the IM enzymes can be further improved by the introduction of short PEG spacers between the BG group and the bead surface (Scheme 1, blue sphere chain). The PEG spacers would create a greater separation between the enzyme and the bead surface and thus limit the interference of the solid support. To test these hypotheses, we varied the length of the surface coating PEG amines as well as of the PEG spacers between the BG and NH2 moieties (see experimental details in the Supporting Information). Table S3 lists the different conditions used to prepare T4 pol, T4 PNK and Taq pol IM enzymes. The effects of PEG coating and PEG spacers on the activity of IM T4 pol is exemplified in Figure 1. T4 pol immobilized on BG beads coated with PEG 750 (IM T4 pol_BGNH2/PEG 750) fully converted substrates into products, indicating a significant improvement in enzyme activity over BG beads coated with diethanolamine. We also noticed that alter-
ing the length of the PEG coating reagent (from 750 to 5000 Da average molecular weight) had little effect on the enzymatic activity of T4 pol (Figures S1 and S2). Interestingly, T4 pol immobilized on beads featuring BG attached through PEG spacers (e.g., BG-PEG4-NH2, BG-PEG12-NH2 and BG-PEG36NH2) showed slightly lower activity than of BG-NH2 with no spacer (Figures 1 & S1). The same trend was observed for IM T4 pol with PEG coating only (e.g., IM T4 pol_BGNH2/PEG 750) when compared to beads with both PEG coating and BG PEG spacers (e.g., IM T4 pol_BG-PEG4NH2/PEG 750) (Figure 1). The decrease in activity could be explained by the possibility of the extended PEG linker, to which the enzyme is covalently conjugated, to fold back towards the bead surface heightening nonproductive interactions with the supporting material. In fact, we found that for IM T4 pol, longer PEG spacers between the BG moiety and the surface reactive NH2 group correlated with increased loss of enzyme activity (BG-PEG4-NH2>BG-PEG12-NH2>BG-PEG36NH2, Figures S1 and S2). It is important to note that the SNAP-tag itself could be regarded as a spacer between the associated enzyme and the bead surface, therefore minimizing the need for an additional PEG spacer at the site of conjugation. Overall, these results demonstrated that the use of PEG coating, and to a lesser extent, a BG spacer with an optimal chain length, can be effective at improving the activity of immobilized DNA replication enzymes such as T4 pol. We also compared the thermostability of soluble and immobilized T4 pol using the same substrates as above to evaluate any retaining enzyme activities after heating at different temperatures. No significant differences in activity were observed after pre-incubation at 20 °C or 35 °C for 30 minutes (Figure S3). When IM enzymes on PEG surface coating beads were pre-incubated at 45 °C for 30 minutes, only a small activity loss (
