The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5′ terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.

Regulation of specific protein function is of great importance for both research and therapeutic development. Many small or large molecules have been developed to control specific protein function, but there is a lack of a universal approach to regulate the function of any given protein. Herein, we report a general host–guest molecular recognition approach involving modification of the protein functional surfaces with genetically encoded unnatural amino acids bearing guest side chains that can be specifically recognized by cucurbit[7]uril. Using two enzymes and a cytokine as models, we showed that the activity of proteins bearing unnatural amino acid could be turned off by host molecule binding, which blocked its functional binding surface. Protein activity can be switched back by treatment with a competitive guest molecule. Our approach provides a general tool for reversibly regulating protein function through molecular recognition and can be expected to be valuable for studying protein functions.

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small‐molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code. Moreover, we demonstrate that bio‐orthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or a therapeutic antibody with high yields, allowing for subsequent chemical conjugation, which will be useful both for research applications and for the development of homogeneously modified protein therapeutics.

Genetic code expansion (GCE) is a powerful technique for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells, which is achieved through evolved aminoacyl-tRNA synthetase mutants. Stability is important for promoting enzyme evolution, and we found that many of the evolved synthetase mutants have reduced thermostabilities. In this study, we characterized two novel pyrrolysyl-tRNA synthetases (PylRSs) derived from thermophilic archaea: Methanosarcina thermophila (Mt) and Methanosarcina flavescens (Mf). Further study demonstrated that the wild-type PylRSs and several mutants were orthogonal and active in both Escherichia coli and mammalian cells and could thus be used for GCE. Compared with the commonly used M. barkeri PylRS, the wild-type thermophilic PylRSs displayed reduced GCE efficiency; however, some of the mutants, as well as some chimeras, outperformed their mesophilic counterparts in mammalian cell culture at 37 °C. Their better performance could at least partially be attributed to the fact that these thermophilic synthetases exhibit a threshold of enhanced stability against destabilizing mutations to accommodate structurally diverse substrate analogues. These were indicated by the higher melting temperatures (by 3–6 °C) and the higher expression levels that were typically observed for the MtPylRS and MfPylRS mutants relative to the Mb equivalents. Using histone H3 as an example, we demonstrated that one of the thermophilic synthetase mutants promoted the incorporation of multiple acetyl-lysine residues in mammalian cells. The enzymes developed in this study add to the PylRS toolbox and provide potentially better scaffolds for PylRS engineering and evolution, which will be necessary to meet the increasing demands for expanded substrate repertoire with better efficiency and specificity in mammalian systems.

With the advances in the field of expanded genetic code, the application of non-canonical amino acid (ncAA) is considered an effective strategy for protein engineering. However, cumbersome and complicated selection schemes limit the extensive application of this technology in Saccharomyces cerevisiae. To address this issue, a simplified selection scheme with confident results was developed and tested in this study. Based on a mutation library derived from Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS), a logic gate in synthetic biology was used to optimize screening procedures. We found that an “and” gate was more suitable than an “or” gate for isolating aminoacyl-tRNA synthetase from S. cerevisiae. The successful incorporation of O-methyltyrosine (OMeY) proved the utility and efficiency of this new selection scheme. After a round of positive selection, several new OMeY-tRNA synthetase (OMeYRS) mutants were screened, and their incorporation efficiency was improved. Furthermore, we characterized the insertion of several tyrosine analogs into Herceptine Fab and discovered that OMeYRS and its mutants were polyspecific. One of these mutants showed an optimal performance to incorporate different ncAAs into recombinant proteins in S. cerevisiae; this mutant was cloned and transfected into mammalian cells, and the results proved its functionality in HEK293 cells. This study could expand the application of ncAA in S. cerevisiae to construct efficient yeast cell factories for producing natural and synthetic products.

A very recent study published in Nature by Amor et al. identifies the urokinase-type plasminogen activator receptor (uPAR) as a broadly induced specific cell-surface marker during cell senescence, which could be used as a target for chimeric antigen receptor (CAR) T cell to specifically ablate senescent cells in vitro and in vivo. The authors intriguingly show that uPAR-specific CAR T-cell therapy improves the treatment outcome of lung adenocarcinomas and efficiently reduces liver fibrosis in mouse models, thus offering a promising novel therapeutic strategy for senescence-associated diseases.

The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. We report a Cas9-RNA conjugation strategy to afford minimal crRNA containing only the guide sequence for the target gene, which may simplify and reduce the cost for large-scale and high-throughput crRNA synthesis and lead to new insights into the design of CRISPR family complexes for diverse purposes.

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems

The Escherichia coli-derived tyrosyl-tRNA synthetase was the first enzyme engineered for genetic code expansion in a eukaryotic system but can charge only a limited set of structurally simple noncanonical amino acids. In contrast, the thermophilic Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase mutants, used in only a prokaryotic system, can charge a surprisingly large set of structurally diverse ncAAs, due to their remarkable structural ability to tolerate mutations. Inspired by this, we characterized a new class of tyrosyl-tRNA synthetase/tRNATyr pairs from thermophilic bacterium Geobacillus stearothermophilus, which is homologous to the E. coli tyrosyl-tRNA synthetase but with better thermostability. This new pair is both orthogonal in mammalian cells and in Saccharomyces cerevisiae for genetic code expansion and can charge a diverse set of ncAAs with a comparable cellular efficiency, better specificity, and lower background, as compared to those of its E. coli homologue. This thermostable enzyme provides an alternative scaffold for synthetase library screening or evolution to genetically encode more structurally complex ncAAs in eukaryotic cells.

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification. However, recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity. Here we used genetic code expansion technique to introduce a surface exposed self-paired di-thiol functional group into proteins, which can be selectively reduced to afford active thiols. Two compounds containing self-paired disulfides were synthesized, and their genetic incorporations were validated using green fluorescent proteins (GFP). The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody. This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.

In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.

Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways, but identification of unknown PTPs for a given substrate in live cells remain technically challenging. Here, we synthesized a series of tyrosine-based irreversible PTP inhibitors and characterized by site-specific encoding on substrate proteins in cells with an expanded genetic code. By fine-tuning the chemical reactivity, we identified optimal active amino acid probes to covalently cross-link a PTP and its substrate both in vitro and in mammalian cells. Using HER2 as an example, we provide first direct evidence of HER2 Y1023 and SHP2 cross-linking in situ in living human cells. Moreover, proteomic analysis using our approach identified PTP1B as a novel phosphatase for HER2 that specifically dephosphorylated pY1221 position, which may shed light on the puzzle of PTP1B’s role in HER2 positive breast cancer. This novel method provides a useful tool for dissecting tyrosine phosphoregulation in living cells.

Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins.

Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Recent studies have suggested that modulation of two or more signaling pathways can achieve substantial weight loss and glycemic stability. We have developed an approach to the generation of bifunctional antibody agonists that activate leptin receptor and GLP-1 receptor. Leptin was fused into the complementarity determining region 3 loop of the light chain alone, or in combination with exendin-4 (EX4) fused at the N-terminus of the heavy chain of Herceptin. The antibody fusions exhibit similar or increased in vitro activities on their cognate receptors, but 50-100-fold longer circulating half-lives in rodents compared to the corresponding native peptides/proteins. The efficacy of the leptin/EX4 dual antibody fusion on weight loss, especially fat mass loss, was enhanced in ob/ob mice and DIO mice compared to the antibody fusion of either EX4 or leptin alone. This work demonstrates the versatility of this combinatorial fusion strategy for generating dual antibody agonists with long half-lives.

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus .We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope-specific RSV vaccine by grafting a neutralizing epitope (F-epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B-cell epitopes.

The field of therapeutic antibodies has been revolutionized over the past decade, led by the development of novel antibody-modification technologies. Besides the huge success achieved by therapeutic monoclonal antibodies, a diversity of antibody derivatives have emerged with hope to outperform their parental antibodies. Here we review the recent development of methodologies to modify immunoglobulin domains and their therapeutic applications. The innovative genetic and chemical approaches enable novel and controllable modifications on immunoglobulin domains, producing homogeneous therapeutics with new functionalities or enhanced therapeutic profiles. Such therapeutics, including antibody-drug conjugates, bispecific antibodies, and antibody/Fc fusion proteins, have demonstrated great prospects in the treatment of cancer, auto-immune diseases, infectious diseases, and many other disorders.

Here, we report the evolution of an orthogonal amber suppressor pyrrolysyl-tRNA synthetase (PylRS)/tRNA_CUA^Pyl pair that genetically encodes the post-translationally modified amino acid, ε-N-2-hydroxyisobutyryl-lysine (HibK), in bacteria and mammalian cells. HibK is a new type of histone mark that is widely distributed in histone proteins. The ability to site-specifically incorporate HibK into proteins provides a useful tool to probe the biological function of this newly identified post-translational modification.

The bovine antibody BLV1H12, which has an ultralong CDR3H, provides a novel scaffold for engineering new functions into the antibody's variable region. By modifying the β-strand "stalk" of BLV1H12 with sequences derived from natural or synthetic protease inhibitors, we have generated antibodies that inhibit bovine trypsin and human neutrophil elastase (HNE) with low nanomolar affinities. We were also able to generate a humanized variant using a human immunoglobulin scaffold that shares a high degree of homology with BLV1H12. Further optimization yielded a highly selective humanized anti-HNE antibody with sub-nanomolar affinity. This work demonstrates a novel strategy for generating antibodies with potent and selective inhibitory activities against extracellular proteases involved in human disease.

We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology.

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a K_d of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future.

Calcineurin inhibitors such as cyclosporine A and FK506 are effective immunosuppressants but produce severe side effects. Rational modification of a previously reported peptide inhibitor, GPHPVIVITGPHEE (K_D ∼ 500 nM), by replacing the two valine residues with tert-leucine and the C-terminal proline with a cis-proline analogue, gave an improved inhibitor ZIZIT-cisPro, which binds to calcineurin with a K_D value of 2.6 nM and is more resistant to proteolysis.

Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is _L-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7-13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2-5-fold higher than that of nonaarginine (R₉). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities.

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNA^Lys,3, which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents.

One-bead-one-compound (OBOC) libraries provide a powerful tool for drug discovery as well as biomedical research. However, screening a large number of beads/compounds (>1 million) and rank ordering the initial hits (which are covalently attached to a solid support) according to their potencies still post significant technical challenges. In this work, we have integrated some of the latest technical advances from our own as well as other laboratories to develop a general methodology for rapidly screening large OBOC libraries. The methodology has been applied to synthesize and screen a cyclic peptide library that features: (1) spatially segregated beads containing cyclic peptides on the surface layer and linear encoding peptides in their interior; (2) rapid on-bead screening of the library (>1 million) by a multistage procedure (magnetic bead sorting, enzyme-linked assay, and fluorescence based screening); (3) selective release of cyclic peptides from single positive beads for solution-phase determination of their binding affinities; and (4) hit identification by partial Edman degradation/mass spectrometry (PED/MS). Screening of the library against protein phosphatase calcineurin (Cn) identified a series of cyclic peptides that bind to the substrate-docking site for nuclear factor of activated T cells (NFAT) with K_D values of ∼1 μM. Further improvement of the affinity and specificity of these compounds may lead to a new class of immunosuppressive agents that are more selective and therefore less toxic than cyclosporine A and FK506.

Peptidylprolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of _D-pThr-Pip-Nal (where Pip is _L-piperidine-2-carboxylic acid and Nal is _L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity, and the most potent inhibitors had K_D (and K_I) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have antiproliferative activity against the cancer cells.

Prolactin receptor is involved in normal lactation and reproduction; however, excessive prolactin levels can cause various reproductive disorders such as prolactinomas. Small-molecule antagonists against the human prolactin receptor (hPRLr) thus have potential clinical applications and may serve as useful molecular probes in biomedical research. In this work, we synthesized a large, support-bound cyclic peptide library (theoretical diversity of 1.2x10⁷) on 90-μm TentaGel beads and screened it against the extracellular domain of hPRLr. To facilitate hit identification, each TentaGel bead was spatially segregated into outer and inner layers, with a cyclic peptide displayed on the bead surface while the bead interior contained the corresponding linear peptide. The identity of a positive bead was revealed by sequencing the linear encoding peptide within the bead by partial Edman degradation/mass spectrometry. Screening of the library resulted in 20 hits, two of which were selected for further analysis and shown to bind to hPRLr with dissociation constants of 2-3 μM.

A host type non-virus gene delivery carrier, phenanthroline-β-cyclodextrin derivative host molecule, was produced which can be used as molecular probe. Interactions between DZY-1 and DNA were investigated by electrophoresis assay. Hind III enzyme inhibition assay was carried out using DNA condensates induced by host molecules or host-guest molecule complexes to explore their ability to inhibit enzyme digestion. Micro-structure of DNA condensates induced by host molecules and host-guest molecule complexes was observed by scanning electron microscope (SEM). Our work indicates the delivery mechanism of DZY-1 used as a gene delivery carrier and also provides a method to design and produce non-virus gene delivery carriers.

Bridged bis(β-cyclodextrin) 1 with a pyridine-2,6-dicarboxamide linker was synthesized, and its inclusion complexation behavior with some aliphatic oligopeptides was investigated in aqueous buffer solution of pH 2.0 and 7.2 at 25 degrees C by means of circular dichroism, fluorescence, and 2D NMR techniques. The results show that the resulting inclusion complexes of 1 with oligopeptides adopt a cooperative "cyclodextrin-guest-cyclodextrin" sandwich binding mode in a neutral media, but a "guest-linker-cyclodextrin" coinclusion binding mode in an acidic media. These switchable binding modes consequently rationalize the binding ability of bis(β-cyclodextrin) 1 at different pH values; that is, 1 shows the stronger association with oligopeptides in a neutral media. Because of the simultaneous contributions of hydrophobic, hydrogen bond, and electrostatic interactions, bis(β-cyclodextrin) 1 affords length-selectivity up to 4.7 for the Gly-Gly/Gly-Gly-Gly pair at pH 2.0 and sequence-selectivity up to 4.2 for the Gly-Leu/Leu-Gly pair at pH 7.2. These phenomena are discussed from the viewpoint of the size-fit concept and the multipoint recognitions between host and guest.