A mimetic peptide of α2,6-sialyllactose promotes neuritogenesis.

Oxidative stress contributes to the pathogenesis of neurodegenerative diseases. With the aim to find reagents that reduce oxidative stress, a phage display library was screened for peptides mimicking α2,6-sialyllactose (6'-SL), which is known to beneficially influence neural functions. Using Sambucus nigra lectin, which specifically binds to 6'-SL, we screened a phage display library and found a peptide comprising identical sequences of 12 amino acids. Mimetic peptide, reverse peptide and scrambled peptide were tested for inhibition of 6'-SL binding to the lectin. Indeed, lectin binding to 6'-SL was inhibited by the most frequently identified mimetic peptide, but not by the reverse or scrambled peptides, showing that this peptide mimics 6'-SL. Functionally, mimetic peptide, but not the reverse or scrambled peptides, increased viability and expression of neural cell adhesion molecule L1 in SK-N-SH human neuroblastoma cells, and promoted survival and neurite outgrowth of cultured mouse cerebellar granule neurons challenged by H2O2-induced oxidative stress. The combined results indicate that the 6'-SL mimetic peptide promotes neuronal survival and neuritogenesis, thus raising hopes for the treatment of neurodegenerative diseases. This study was approved by the Medical Ethics Committee of Shantou University Medical College, China (approval No. SUMC 2014-004) on February 20, 2014.

Chinese Library Classification No. R453; R741; Q816

Introduction

The mammalian central nervous system is particularly vulnerable to oxidative stress, a phenomenon that is related to epigenetic factors, such as, for instance, low antioxidant levels in an organ with high oxygen consumption, surplus of iron ions, and high polyunsaturated fatty acid content (Ritchie et al., 2003; Farajdokht et al., 2017). Oxidative stress is hence one of the factors that cause neurodegeneration development and progression (Carri et al., 2015; Fischer and Maier, 2015). Oxidative stress-induced neuronal cell death underlies the progression of multiple neural diseases, such as Alzheimer’s disease, schizophrenia and stroke (Hayashi-Takagi et al., 2014; Volpe and Paneni, 2015; Kamat et al., 2016; Chang et al., 2018). It is therefore reasonable to expect that anti-oxidative treatments may be applied as an appropriate strategy to ameliorate the symptoms of neurodegenerative diseases (Xu et al., 2017).

Sialic acid-containing glycans carried by glycoproteins and glycolipids have been broadly applied in clinical treatment of nervous system diseases (Ladeby et al., 2005; Varki, 2008; Du et al., 2009). One of the beneficial sialic acid-containing compounds is sialyllactose (SL), and it is composed of sialic acid and lactose, and classified into α2,3-SL and α2,6-SL dependent on the position at which the sialic acid is attached to lactose (Martin-Sosa et al., 2003). As a major source of sialic acid, SL functions as a component of gangliosides present on neuronal surfaces (Wang and Brand-Miller, 2003; ten Bruggencate et al., 2014), and it is crucial for brain development and function (Charbonneau et al., 2016; Liu et al., 2017). As an essential component of milk, it has been reported to display pro-active neural functions (ten Bruggencate et al., 2014; Tarr et al., 2015; Nagao et al., 2017; Jeon et al., 2018; Kang et al., 2018; Zehra et al., 2018). Difficulties in applying SL clinically relate to the problem that SL is not only cumbersome to purify from biological sources, but also very demanding to produce chemically. It is therefore important to obtain a reagent, such as a peptide, that specifically mimics the beneficial biological activities of SL and can be produced in sufficient amounts.

First introduced by Smith (1985), phage display technology has opened a powerful approach to obtain peptide ligands for almost any target. It can produce up to 1010 diverse peptides or protein fragments (Goldflam and Ullman, 2015; Galán et al., 2016; Messing, 2016). Peptide mimics for the human natural killer glycan (HNK-1) and α2,8-polysialic acid identified by phage display screening (Simon-Haldi et al., 2002; Torregrossa et al., 2004). They have shown to mimic the function of their glycan counterparts, with increased stability, added benefits of ease of production, and reduced generation cost (Ernst and Magnani, 2009; Magnani and Ernst, 2009).

The aim of this study was to use novel ways for identifying molecules that ameliorate oxidative stress. We investigated whether with the phage display method peptides can be identified that mimic the structure and function of 6′-SL and whether the selected mimetic peptide can protect cultured neurons against oxidative stress.

Materials and Methods

Animals

Twenty C57BL/6 specific-pathogen-free (SPF) female mice and ten C57BL/6 SPF male mice at the age of 4 weeks for the breeding of mice for primary culture of cerebellar granule neurons were obtained from the Guangdong Medical Laboratory Animal Center (licence No. SCXK (Yue) 2008-0002) and maintained on a 12-hour light/12-hour dark cycle at 25°C, with food and water ad libitum. All experimental protocols were approved by the Medical Ethics Committee of Shantou University Medical College, China (approval No. SUMC 2014-004) on February 20, 2014. Animals were maintained according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal research (Kilkenny et al., 2010).

Screening for isolation of a mimetic peptide for 6′-SL

We used the Ph.D.TM−12 phage display peptide library (PDPL, New England BioLabs, Ipswich, MA, USA) where peptides are presented by the pIII protein on the pili of bacteriophage M13. Screening was performed as described (Simon-Haldi et al., 2002; Loers et al., 2019) with minor modifications. Briefly, heparin-binding 96-well plate wells (Corning, New York, USA) were coated overnight at 4°C with 15 μL of 1 mg/mL Sambucus nigra lectin (Cat# B-1305, Vector Laboratories, Burlingame, CA, USA) diluted in 135 μL NaHCO3 (0.1 M, pH 8.6) to give a final concentration of 100 μg/mL. After blocking overnight at 4°C with 1% bovine serum albumin in phosphate-buffered saline (PBS, pH 7.3), the wells were then incubated with 1.5 × 1011 phages in Tris-buffered saline containing 0.1% Tween for 1 hour at room temperature. Wells were washed 10 times with Tris-buffered saline (pH 7.4) containing 0.1%, 0.3%, and 0.5% (v/v) Tween-20 (Cat# 9005-64-5, Sigma-Aldrich, St. Louis, MO, USA) in the first, second, and third rounds of panning, respectively. Phages were eluted using 100 μL of 1 M 6′-SL (Cat# 35890-39-2, Dextra Laboratories, Reading, England, UK) in each round of panning. After the first round of panning, eluted phages were amplified, and 2 × 1011 phages were taken for panning in the second and third rounds. Individual phage clones derived from the 3rd round of screening were isolated, and single-stranded phage DNA was purified for DNA sequencing (Sangon Biotech, Shanghai, China) to determine the peptide sequences. The following sequence was found in three phage clones: (H-PRHGKKPTNKRK-OH). The mimetic peptide, reverse peptide (H-KRKNTPKKGHRP-OH) and scrambled peptide (H-RPGHKKTPKNKR-OH) were synthesized by Hanhong Biochemical Company (Shanghai, China).

Phage binding enzyme-linked immunosorbent assay

To confirm that the obtained phages express a peptide mimicking 6′-SL, wells were pre-coated overnight at 4°C with 100 μL of 100 μg/mL Sambucus nigra lectin or NaHCO3 as vehicle control. After blocking with 100 μL of 1% bovine serum albumin in PBS for 1 hour at room temperature, the wells were incubated with 1 × 1010 purified phages in PBS for 2 hours at room temperature, with PBS as vehicle control. Wells were washed 6 times with Tris-buffered saline containing 0.1% Tween-20 before incubation with 100 μL of horseradish peroxidase-conjugated M13 antibodies (Cat# 27-9421-01, GE Healthcare, Piscataway, NJ, USA) in blocking solution for 1 hour at room temperature. The wells were then washed 6 times with Tris-buffered saline containing 0.03% Tween 20, and with 100 μL of 2,2′-azinobis-(3-et hylbenzthiazoline-6-sulphonate) (Cat# 30931-67-0, Sigma-Aldrich) finally. Absorbance was determined at 415 nm in a microtiter plate reader (Tecan Infinite® M1000 Pro, Tecan, Switzerland).

Competition enzyme-linked immunosorbent assay

To investigate whether the mimetic peptide can mimic 6′-SL, the mimetic or scrambled peptides were coated in 96-well plates at 20 μg/mL in PBS overnight at 4°C, and thereafter blocked with Carbo-free™ Blocking Solution (Cat# SP-5040, Vector Laboratories) for 2 hours at room temperature. After washing 5 times for 5 minutes each with PBS containing 0.1% Tween-20, wells were incubated overnight at 4°C with different dilutions (1:20, 1:100, 1:200, 1:500) of a 6′-SL/biotinylated Sambucus nigra lectin stock solution (Cat# B-1305-2, Vector Laboratories). After washing 5 times with PBS containing 0.1% Tween-20, wells were incubated with 100 μL horseradish peroxidase-coupled streptavidin (1:3000, Cat# BA1088, Boster, Wuhan, China) for 1 hour at 37°C. After washing 6 times with 0.1% PBS containing 0.1% Tween-20 for 5 minutes each at room temperature, wells were incubated with 2,2′-azinobis-(3-et hylbenzthiazoline-6-sulphonate) chromogenic substrate solution Substrate Chromogenic Solution (Cat# 37615, Thermofisher Scientific, Waltham, MA, USA) containing 30% H2O2 for 30 minutes at room temperature. Absorbance was determined with an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm (Tecan Infinite® M1000 Pro).

Culture of SK-N-SH cells

Human neuroblastoma SK-N-SH cells were purchased from Chinese Type Culture Collection and cultured as described (Liu et al., 2013). Cells were treated with H2O2 as described (Zhang et al., 2017) with minor modifications. Briefly, 1 × 104 human neuroblastoma SK-N-SH cells were seeded into 96-well tissue culture plates (for cell viability assay) or 24-well tissue culture plates (for immunofluorescence staining of L1). Cells in wells of tissue culture plates were pretreated with 250 μM H2O2 for 2 hours prior to a 48-hour treatment with peptides at 20 μg/mL, when cell viability assay and L1 immunostaining were performed.

Culture of mouse cerebellar granule neurons

Cerebellar granule neurons were primary cultured from mice as described (Loers et al., 2019). Briefly, dissociated cells (2 × 104 cells in 100 μL Neurobasal A medium without B-27) were seeded into poly-D-lysine-coated 96-well tissue culture plates (for cell viability assay) or 24-well tissue culture plates (for neurite outgrowth assay). Cells were then pre-treated with 250 μM H2O2 for 2 hours prior to the 24-hour treatment with peptides at 20 μg/mL.

Cell viability assay

We evaluated cell viability using a Cell Counting Kit (CCK-8; Cat# HY-K0301, MedChem Express, China). Briefly, 10 μL CCK-8 solution was added to each well, respectively, and incubated at 37°C for 2 hours. Absorbance was then measured at 490 nm in a multi-well plate reader (Tecan Infinite® M1000 Pro).

Neurite outgrowth assay

Cells were fixed by adding 100 μL of 4% formaldehyde in PBS and then stained with Crystal Violet Staining Solution (Cat# C8470, Solarbio, Beijing, China). Neurite lengths of individual cerebellar granule neurons were measured at a 40× magnification by an Image Analysis System (IX-51, Olympus, Tokyo, Japan). Only neurites with a length of at least one diameter of the cell body and without contact with other cells were measured. Approximately 50 cells were counted per experiment and per experimental group, and each experiment was repeated for three times.

Immunofluorescence staining

After rinsing with PBS, human neuroblastoma SK-N-SH cells were fixed with 4% formaldehyde in PBS for 10 minutes, and then washed with PBS. 10% normal donkey serum was used to block the unspecific binding sites (Cat# 017-000-001, Jackson ImmunoResearch Laboratories) in PBS at room temperature for 40 minutes. Cells were then incubated with mouse monoclonal antibody against L1 (1:200; Cat# No. MAB777, R&D Systems, Minneapolis, MN, USA) overnight at 4°C. Samples were rinsed three times with PBS for 5 minutes each at room temperature and incubated with donkey anti-mouse secondary antibody conjugated to Alexa Fluor® 488 (1:500, Cat# A28175, Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. The coverslips were mounted using the ProLong® Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (Cat# P36935, Gibco; ThermoFisher Scientific). Confocal images were acquired using the FV10-ASW software (Ver. 01.01) under an Olympus confocal system (FV-1000, Olympus, Tokyo, Japan). The immunofluorescence intensity indicating the level of L1 was measured using ImageJ 5.0.

Statistical analysis

GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analyses. Data are expressed as means ± SEM. One-way analysis of variance was performed followed by the post-hoc Bonferroni test, and P < 0.05 was considered statistically significant.

Results

Identification of a mimetic peptide

To identify a mimetic peptide, a phage display library containing 1 × 109 different filamentous phages presenting 12-mer peptides on phage coat protein pIII was screened. The eluted phage clones were probed for their binding to the 6′-SL-specific Sambucus nigra lectin by ELISA, with the phage clones M1, M5, M12 and M14 clones showing best binding (). Clones were sequenced and found to contain identical insert sequences, except for M5 which failed in sequencing ().

Identification of phages binding to Sambucus nigra lectin.

After three panning rounds, duplicate samples of phages M1, M5, M12, and M14 were identified to bind to lectin with high affinity by enzyme-linked immunosorbent assays (black bars). Vehicle controls, also in duplicate, are indicated by white bars.

To verify mimic specificity for 6′-SL, the peptides with the best binding in the phage ELISA were synthesized. Their reverse and scrambled counterparts were also synthesized. With increasing ratios of 6′-SL over biotinylated Sambucus nigra lectin in the pre-incubation solution, the binding of the mimetic peptide to the lectin was competitively decreased, indicating that the mimetic peptide was structurally similar to 6′-SL. The scrambled peptide did not show any binding in comparison to the blank control groups ().

Enzyme-linked immunosorbent assay showing concentration-dependent inhibition of biotinylated Sambucus nigra (Bio-SNA) binding to α2,6-sialyllactose (6′-SL) mimetic peptide-coated substrate by 6′-SL.

Negative controls were substrate coated with scrambled peptide, bovine serum albumin (BSA) and phosphate-buffered saline (PBS). Data are expressed as the mean ± SEM. **P < 0.01 (one-way analysis of variance followed by the Bonferroni post-hoc test). The experiment was repeated three times.

Mimetic peptide increases the viability of H2O2-stressed SK-N-SH cells and cerebellar granule neurons

To explore the effect of the mimetic peptide on neuronal survival in vitro, cell viability assays were performed with SK-N-SH cells or cerebellar granule neurons after pre-treatment with 250 μM H2O2 for 2 hours before adding the mimetic, reverse or scrambled peptides each at a concentration of 20 μg/mL for 48 hours or 24 hours. In comparison with the vehicle control group, H2O2 treatment reduced survival of SK-N-SH cells. The mimetic peptide increased survival of H2O2 stressed SK-N-SH cells, whereas the reverse and scrambled peptides did not decrease cell death (). A similar result was observed for viability of cerebellar granule neurons ().

The α2,6-sialyllactose (6′-SL) mimetic peptide, but not its reverse or scrambled counterparts, enhances survival of SK-N-SH cells (A) and mouse cerebellar granule neurons (B) under H2O2-induced oxidative stress.

6′-SL mimetic peptide: H-PRHGKKPTNKRK-OH; 6′-SL reverse peptide: H-KRKNTPKKGHRP-OH; 6′-SL scrambled peptide: H-RPGHKKTPKNKR-OH. Data are expressed as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (one-way analysis of variance followed by the Bonferroni post-hoc test). The experiment was repeated five times.

Mimetic peptide increases neurite length of H2O2-stressed cerebellar granule cells

To verify the effects of the mimetic peptide on neurite outgrowth under oxidative stress, neurite outgrowth of cerebellar granule cells was determined. In comparison with the vehicle control group, H2O2 treatment reduced the lengths of neurites. The mimetic peptide increased neurite lengths compared with the H2O2-treated group, whereas the reverse and scrambled peptides did not increase neurite lengths ().

The α2,6-sialyllactose (6′-SL) mimetic peptide, but not its reverse or scrambled counterparts, enhances neurite outgrowth of mouse cerebellar granule neurons under H2O2-induced oxidative stress.

(A) Representative images of cerebellar granule neurons with different neurite lengths (arrows). The mimetic peptide increased neurite lengths compared with the H2O2-treated group, whereas the reverse and scrambled peptides did not increase neurite lengths. Scale bar: 20 µm. (B) Quantification of cerebellar granule neuron neurite lengths. Data are expressed as the mean ± SEM. **P < 0.01 (one-way analysis of variance followed by the Bonferroni post-hoc test). The experiment was repeated three times.

Mimetic peptide increases L1 expression in H2O2-stressed SK-N-SH cells

To study the effects of the mimetic peptide on the expression of the neural cell adhesion molecule L1 under oxidative stress, we performed immunofluorescence staining on SK-N-SH cells. In comparison with the control group, H2O2 treatment reduced L1 expression. Compared with the H2O2-treated group, the mimetic peptide increased L1 expression under H2O2 conditions, whereas L1 levels did not change after treatment with the reverse or scrambled peptides ().

The α2,6-sialyllactose (6′-SL) mimetic peptide enhances L1 immunopositivity in SK-N-SH cells under H2O2-induced oxidative stress.

(A) Representative images of L1 immunofluorescence staining in H2O2-stressed SK-N-SH cells. The mimetic peptide increased L1 expression under H2O2 conditions, whereas L1 levels did not change after treatment with the reverse or scrambled peptides. Scale bar: 20 µm. (B) Quantification of relative L1 immunopositivity in H2O2-stressed SK-N-SH cells. Data are expressed as the mean ± SEM. ***P < 0.001 (one-way analysis of variance followed by the Bonferroni post-hoc test). The experiment was repeated five times.

Discussion

Recent studies have verified the potential of mimetic peptides for drug design and therapy (Mehanna et al., 2010; Masand et al., 2012; Prost et al., 2012). Screening of phage display libraries benefits many molecular and cellular functions, as evidenced by peptide drug discovery (Hamzeh-Mivehroud et al., 2013), identification of biomarkers (Ghoshal et al., 2016), isolation of high-affinity antibodies (Azzazy and Highsmith, 2002), and vaccine development (Aghebati-Maleki et al., 2016). Regarding the mimicry of pro-active carbohydrates, mimetic peptides for α2,8-polysialic acid promote functional recovery following spinal cord injury in mice (Marino et al., 2009; Mehanna et al., 2010) and accelerate myelination and functional recovery following peripheral nerve injury in the mouse (Mehanna et al., 2009). In the present study, we found a peptide that mimics not only the structure of 6′-SL, but enhances neuronal survival and neuritogenesis, accompanied by increased expression of the regeneration-beneficial cell adhesion molecule L1.

Previous studies have shown degenerative changes in the cerebellum in Huntington’s disease, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (Colloby et al., 2014; Tan et al., 2014; Burciu et al., 2015; Wolf et al., 2015). Thus, cerebellar granule neurons have been widely used to study apoptosis in neurodegenerative diseases (Chen et al., 2009), since they are particularly vulnerable to oxidative insults due to their membranes being mainly composed of polyunsaturated fatty acids, which are substrates for reactive oxygen species (Wang et al., 2017). Neuroblastoma cells have also served for studying neural functions because of their similarity to mature neural stem cells that can be differentiated into neurons and astrocytes (Ross et al., 2003; Ross and Spengler, 2007). Also, they have been paradigmatic for the study of neurons in cell culture. In a previous study, the cell viability of SK-N-SH cells was decreased in a dose- and time-dependent manner in response to the treatment of H2O2 (Zhang et al., 2017). In our present research, we treated the SK-N-SH cells and cerebellar granule neurons with 250 μM H2O2 for only 2 hours to induce mild cell death, which is typical to early-onset neurodegenerative diseases. Indeed, both human neuroblastoma and mouse cerebellar granule neurons responded favorably to the 6’-SL mimetic peptide in terms of cell survival.

Neurite outgrowth is an important feature of neuronal plasticity and regeneration after injury (Khodagholi et al., 2012). Previous studies have reported that a tenascin-C mimetic peptide-coupled nanofiber gel can promote neurite outgrowth and cell migration of neurosphere-derived cells (Berns et al., 2016). A mimetic peptide of the S-100 protein attenuates the progression of neuropathy and enhances peripheral nerve regeneration in myelin protein P0 null mice (Moldovan et al., 2013). A peptide mimetic targeting the neural cell adhesion molecule NCAM can promote neural plasticity and spatial learning in the hippocampus (Kraev et al., 2011). Neuroplastin-65 and a mimetic peptide derivative of its homophilic binding site can modulate neuritogenesis and neuroplasticity (Owczarek et al., 2011). Phage display libraries have also been useful in selecting peptides that bind to targets of choice: chondroitin-4-sulfate-binding peptides enhance neurite outgrowth on inhibitory chondroitin sulfate proteoglycans (Loers et al., 2019).

We used the neural cell adhesion molecule L1 as an indicator for beneficial cell functions, because it plays a pivotal role in neural cell-to-cell interactions (Lindner et al., 1983; Faissner et al., 1984; Rathjen and Schachner, 1984) by supporting neuronal survival and migration, neurite outgrowth, axon guidance, myelination (Wood et al., 1990; Maness and Schachner, 2007; Sytnyk et al., 2017) and synaptic plasticity (Lüthi et al., 1994). Persistent expression of L1 is crucial for learning, memory, and regeneration following injury (Liljelund et al., 1994; Chaisuksunt et al., 2000; Zhang et al., 2005). L1 overexpression ameliorates the abnormal phenotypes in mouse models of neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease (Cui et al., 2010; Djogo et al., 2013). Under these auspices, we were able to show in the present study that the 6′-SL mimetic peptide increases L1 expression.

Taken together, our results reveal a neuroprotective role of the mimetic peptide for 6’-SL under oxidative stress conditions. Further studies are needed to identify the influence of the mimetic peptide in central nervous system injury, which are encouraged by the experience that adhesion molecules showing pro-activity in vitro can be pro-active in vivo. Also, it will be important to identify the downstream signaling molecules related to the neuroprotective effects of the mimetic peptide under pathological conditions with the hope that the mimetic peptide for α2,6-sialyllactose may be a novel candidate for therapy of neurodegenerative diseases. In vivo studies using animal models of neurodegenerative diseases may be needed to further identify the therapeutic efficacy of this peptide in the future. In conclusion, a mimetic peptide for 6′-SL with high affinity to Sambucus nigra lectin was identified by phage binding and competition ELISA. This peptide supports beneficial functions in cultured neurons, providing hopes for a peptide-based therapy to ameliorate the consequences of oxidative stress in neurodegenerative diseases in a clinical setting.

Additional file: Open peer review reports .

Table 1

Sequences of phage-encoded peptides that bound to Sambucus nigra lectin

Clone names	Codons (5′–3′)	Peptide sequences (N′–C′)
M10	GCT GAT GCT AGG CCG TGG TGG AAG AGT CAG GGT TTT	ADARPWWKSQGF
M9	TTT CCG TAT CCT ATG AAT AAG CAG ACT AAT GGT ACT	FPYPMNKQTNGT
M2	AAT CAT CGG AAG GTT AGT AGG CAT GCG ACT CAT TTT	NHRKVSRHATHF
M6	AAT CAT CGG AAG GTT AGT AGG CAT GCG ACT CAT TTT	NHRKVSRHATHF
M8	AAT CAT CGG AAG GTT AGT AGG CAT GCG ACT CAT TTT	NHRKVSRHATHF
M13	AAT CAT CGG AAG GTT AGT AGG CAT GCG ACT CAT TTT	NHRKVSRHATHF
M1	CCG AGG CAT GGG AAG AAG CCT ACG AAT AAG AGG AAG	PRHGKKPTNKRK
M12	CCG AGG CAT GGG AAG AAG CCT ACG AAT AAG AGG AAG	PRHGKKPTNKRK
M14	CCG AGG CAT GGG AAG AAG CCT ACG AAT AAG AGG AAG	PRHGKKPTNKRK
M7	ACG GTT GAT TCG GCT AGT CTT TTG CAG AGT CGT ACT	TVDSASLLQSRT
M3	TGG GGG TTT CAT TGG CCG GTG TAT CCT CCG TCT AGG	WGFHWPVYPPSR
M4	Empty carrier
M11	Empty carrier
M5	Failed to be sequenced