RESUMO
A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.
Assuntos
Teste para COVID-19/métodos , COVID-19/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/diagnóstico , Humanos , Transcrição Reversa , SARS-CoV-2/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively underpin and rationalise the BDP approach to point variant sensing, which relies on different responses to duplex formation rather than different duplex binding strengths.
Assuntos
Antracenos/química , DNA/química , DNA/genética , Sondas Moleculares/química , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Coloração e RotulagemRESUMO
It is a challenge within the field of biomimetics to recreate the properties of light-harvesting antennae found in plants and photosynthetic bacteria. Attempts to recreate these biological structures typically rely on the alignment of fluorescent moieties via attachment to an inert linear scaffold, e.g. DNA, RNA or amyloid fibrils, to enable Förster resonance energy transfer (FRET) between attached chromophores. While there has been some success in this approach, refinement of the alignment of the chromophores is often limited, which may limit the efficiency of energy transfer achieved. Here we demonstrate how linear dichroism spectroscopy may be used to ascertain the overall alignment of chromophores bound to the M13 bacteriophage, a model linear scaffold, and demonstrate how this may be used to distinguish between lack of FRET efficiency due to chromophore separation, and chromophore misalignment. This approach will allow the refinement of artificial light-harvesting antennae in a directed fashion.
RESUMO
DNA three-way junctions (3WJs) are essential structural motifs for DNA nanoarchitectures and DNA-based materials. We report herein a metal-responsive structural transformation between DNA duplexes and 3WJs using artificial oligonucleotides modified with a 2,2'-bipyridine (bpy) ligand. A mixture of bpy-modified DNA strands and natural complementary strands were self-assembled exclusively into duplexes without any transition metal ions, while they formed 3WJs in the presence of NiII ions. This transformation was induced by the formation of an interstrand NiII(bpy)3 complex, which served as a template for the 3WJ assembly. Altering the amount and identity of the metal ion regulated the 3WJ induction efficiency. Removal of the metal using EDTA quantitatively regenerated the duplexes. The metal-dependent structural conversion shown here has many potential applications in the development of stimuli-responsive DNA materials.
RESUMO
The direct incorporation of macrocyclic cyclidene complexes into DNA via automated synthesis results in a new family of metal-functionalized DNA derivatives that readily demonstrate their utility through the ability of one redox-active copper(II)-containing strand to distinguish electrochemically between all four canonical DNA nucleobases at a single site within a target sequence of DNA.
Assuntos
DNA/química , Técnicas Eletroquímicas , Compostos Macrocíclicos/química , Nucleotídeos/análise , Compostos Organometálicos/química , Estrutura MolecularRESUMO
The ability to discriminate between epigenetic variants in DNA is a necessary tool if we are to increase our understanding of the roles that they play in various biological processes and medical conditions. Herein, it is demonstrated how a simple two-step fluorescent probe assay can be used to differentiate all three major epigenetic variants of cytosine at a single locus site in a target strand of DNA.
Assuntos
5-Metilcitosina/química , Antracenos/química , Citosina/análogos & derivados , Citosina/química , DNA/química , Corantes Fluorescentes/química , Conformação de Ácido NucleicoRESUMO
Modified DNA strands undergo a reversible light-induced reaction involving the intramolecular photodimerization of two appended anthracene tags. The photodimers exhibit markedly different binding behavior toward a complementary strand that depends on the number of bases between the modified positions. By preforming the duplex, photochromism can be suppressed, illustrating dual-mode gated behavior.
Assuntos
Antracenos/química , DNA/efeitos da radiação , Antracenos/efeitos da radiação , Sequência de Bases , DNA/química , LuzRESUMO
Single nucleotide polymorphisms within a sequence of a gene associated with prostate cancer were identified using oligodeoxynucleotide probe sequences bearing internal anthracene fluorophores proximal to the SNP site. Depending upon the nature of the synthesised target sequences, probe-target duplex formation could lead to enhanced or attenuated fluorescence emission from the anthracene, enabling detection of a proximal base-pair as either matching or mismatching.
Assuntos
Sondas de DNA/química , DNA/química , Corantes Fluorescentes/química , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/metabolismo , Antracenos/química , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Humanos , Masculino , Modelos Químicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Neoplasias da Próstata/genética , Espectrofotometria Ultravioleta , Temperatura , TermodinâmicaRESUMO
A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.
Assuntos
Citosina/química , DNA/química , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico/métodos , Antracenos/química , Dicroísmo Circular , Metilação de DNA , Sondas de DNA/química , Espectrometria de FluorescênciaRESUMO
Monoamine oxidase (MAO) is an important drug target for the treatment of neurological disorders. A series of indole and benzofuran derivatives were synthesised and evaluated as inhibitors of the two MAO isoforms, MAO-A and MAO-B. In general, the derivatives were found to be selective MAO-B inhibitors with K(i) values in the nanoMolar (nM) to microMolar (microM) concentration range. The most potent MAO-B inhibitor, 3,4-dichloro-N-(2-methyl-1H-indol-5-yl)benzamide, exhibited a K(i) value of 0.03 microM and was 99 fold more selective for the B isoform. We conclude that these indole and benzofuran derivatives are promising reversible MAO-B inhibitors with a possible role in the treatment of neurodegenerative diseases such as Parkinson's disease (PD).
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Indóis/química , Indóis/farmacologia , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Benzofuranos/síntese química , Humanos , Indóis/síntese química , Modelos Moleculares , Inibidores da Monoaminoxidase/síntese química , Doença de Parkinson/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Monoamine oxidase B (MAO-B) and nitric oxide synthase (NOS) have both been implicated in the pathology of neurodegenerative diseases. In an attempt to design dual-target-directed drugs that inhibit both these enzymes, a series of pteridine-2,4-dione analogues were synthesised. The compounds were found to be relatively weak NOS inhibitors but showed promising MAO-B activity with 6-amino-5-[(E)-3-(3-chloro-phenyl)-prop-2-en-(E)-ylideneamino]-1,3-dimethyl-1H-pyrimidine-2,4-dione and 6-[(E)-2-(3-chloro-phenyl)-vinyl]-1,3-dimethyl-1H-pteridine-2,4-dione inhibiting MAO-B with IC(50) values of 0.602 and 0.314 microM, respectively. The pteridine-2,4-dione analogues thus show promise as scaffolds for the development of potent reversible MAO-B inhibitors and as observed in earlier studies, the most potent inhibitors were obtained with 3-chlorostyryl substitution.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores da Monoaminoxidase/síntese química , Monoaminoxidase/química , Fármacos Neuroprotetores/síntese química , Óxido Nítrico Sintase/antagonistas & inibidores , Pteridinas/síntese química , Animais , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Conformação Molecular , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase/metabolismo , Papio , Pteridinas/química , Pteridinas/farmacologiaRESUMO
The blood-brain barrier is formed by the brain capillary endothelium and plays the predominant role in controlling the passage of substances between the blood and the brain. Recent studies on polycyclic structures, i.e. pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane and amantadine, indicated favourable distribution thereof to the brain and it was concluded that these polycyclic structures and their derivatives penetrate the blood-brain barrier readily. A series of novel polycyclic prodrugs incorporating the well known non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid and ibuprofen, were synthesised and screened for blood-brain barrier permeability and antioxidant activity. Increased levels of both NSAIDs were detected in the brain tissue of C57BL/6 mice after administration of the synthesised prodrugs, indicating favourable blood-brain barrier permeation. Results from a lipid peroxidation assay indicated that the ester and amide prodrugs significantly increased the ability of the drugs to attenuate lipid peroxidation. These novel prodrugs thus readily penetrate the blood-brain barrier and exhibit increased antioxidant activity when compared to the free NSAIDs.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Ibuprofeno/farmacologia , Fármacos Neuroprotetores/farmacologia , Compostos Policíclicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Antioxidantes/síntese química , Antioxidantes/química , Antioxidantes/farmacologia , Aspirina/síntese química , Aspirina/química , Barreira Hematoencefálica/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ibuprofeno/síntese química , Ibuprofeno/química , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Permeabilidade/efeitos dos fármacos , Compostos Policíclicos/síntese química , Compostos Policíclicos/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologiaRESUMO
The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.
Assuntos
Proteínas Aviárias , Proteínas de Transporte/química , Músculo Liso/citologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Transporte/fisiologia , Diferenciação Celular , Galinhas , Cristalografia por Raios X , Proteínas com Domínio LIM , Modelos Moleculares , Dados de Sequência Molecular , Músculo Liso/química , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Dedos de Zinco/fisiologiaRESUMO
Members of the cysteine-rich protein (CRP) family are evolutionarily conserved proteins that have been implicated in the processes of cell proliferation and differentiation. In particular, one CRP family member has been shown to be an essential regulator of cardiac and skeletal muscle development. Each of the three vertebrate CRP isoforms characterized to date is composed of two copies of the zinc-binding LIM domain with associated glycine-rich repeats. In this study, we have addressed the biological significance of the CRP multigene family by comparing the subcellular distributions, biochemical properties, and expression patterns of CRP1, CRP2, and CRP3/MLP. Our data reveal that all three CRP family members, when expressed in adherent fibroblasts, associate specifically with the actin cytoskeleton. Moreover, all three CRP isoforms are capable of interacting with the cytoskeletal proteins alpha-actinin and zyxin. Together, these observations suggest that CRP family members may exhibit overlapping cellular functions. Differences between the three CRPs are evident in their protein expression patterns in chick embryos. CRP1 expression is detected in a variety of organs enriched in smooth muscle. CRP2 is restricted to arteries and fibroblasts. CRP3/MLP is dominant in organs enriched in striated muscle. CRP isoform expression is also developmentally regulated in the chick. Our findings suggest that the three CRP family members perform similar functions in different muscle derivatives. The demonstration that all members of the CRP family are associated with cytoskeletal components that have been implicated in the assembly and organization of filamentous actin suggests that CRPs contribute to muscle cell differentiation via effects on cytoarchitecture.
Assuntos
Cisteína , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Variação Genética , Zíper de Leucina , Proteínas Nucleares/química , Fatores de Transcrição/química , Actinas/análise , Sequência de Aminoácidos , Animais , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT , Embrião de Galinha , Galinhas , Sequência Conservada , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Fibroblastos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/biossíntese , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is alpha-actinin. We have shown that the CRP1-alpha-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-alpha-actinin interaction is 1.8 +/- 0.3 microM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and alpha-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that alpha-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin-binding domain of alpha-actinin. In reciprocal mapping studies, we showed that alpha-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the alpha-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the alpha-actinin-binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with alpha-actinin may be critical for its role in muscle differentiation.
Assuntos
Actinina/fisiologia , Proteínas Aviárias , Proteínas de Transporte/fisiologia , Proteínas de Homeodomínio/fisiologia , Músculo Liso/metabolismo , Citoesqueleto de Actina/metabolismo , Actinina/isolamento & purificação , Actinina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Galinhas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas com Domínio LIM , Músculo Liso/citologia , Músculo Liso/fisiologia , Ligação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Frações Subcelulares/metabolismoRESUMO
Zyxin is a component of adhesion plaques that has been suggested to perform regulatory functions at these specialized regions of the plasma membrane. Here we describe the isolation and characterization of cDNAs encoding human and mouse zyxin. Both the human and mouse zyxin proteins display a collection of proline-rich sequences as well as three copies of the LIM domain, a zinc finger domain found in many signaling molecules. The human zyxin protein is closely related in sequence to proteins implicated in benign tumorigenesis and steroid receptor binding. Antibodies raised against human zyxin recognize an 84-kDa protein by Western immunoblot analysis. The protein is localized at focal contacts in adherent erythroleukemia cells. By Northern analysis, we show that zyxin is widely expressed in human tissues. The zyxin gene maps to human chromosome 7q32-q36.
Assuntos
Adesão Celular , Metaloproteínas/química , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Mapeamento Cromossômico , Proteínas do Citoesqueleto , DNA Complementar/química , DNA Complementar/isolamento & purificação , Glicoproteínas , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , ZixinaRESUMO
LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha-helix appears to be packed more tightly against the preceding antiparallel beta-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.
Assuntos
Proteínas de Transporte/química , Proteínas , Sequência de Aminoácidos , Animais , Proteínas com Domínio LIM , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proto-Oncogene Mas , RatosRESUMO
The three dimensional solution structure of the carboxy terminal LIM domain of the avian Cysteine Rich Protein (CRP) has been determined by nuclear magnetic resonance spectroscopy. The domain contains two zinc atoms bound independently in CCHC (C = Cys, H = His) and CCCC modules. Both modules contain two orthogonally-arranged antiparallel beta-sheets, and the CCCC module contains an alpha-helix at its C terminus. The modules pack due to hydrophobic interactions forming a novel global fold. The structure of the C-terminal CCCC module is essentially identical to that observed for the DNA-interactive CCCC modules of the GATA-1 and steroid hormone receptor DNA binding domains, raising the possibility that the LIM motif may have a DNA binding function.
Assuntos
Proteínas Aviárias , Modelos Moleculares , Proteínas Musculares/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/química , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sítios de Ligação , Galinhas , DNA/metabolismo , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência MolecularRESUMO
Site-directed mutagenesis was carried out to map the residues that form the two Zn(II) sites within a LIM domain. The C-terminal LIM domain derived from the cysteine-rich protein was utilized for this analysis and is referred to as LIM2. Seven cysteinyl residues and a single histidyl residue in the LIM2 sequence, CX2CX17HX2CX2CX2CX17CX2C, comprise the conserved residues in the LIM consensus that are potential Zn(II) ligands. Two Zn(II) binding sites exhibiting tetrathiolate (S4) and S3N1 Zn(II) coordination are displayed by LIM2 (Kosa, J. L., Michelsen, J. W., Louis, H. A., Olsen, J. I., Davis, D. R., Beckerle, M. C., and Winge, D. R. (1994) Biochemistry 33, 468-477). Site-directed mutagenesis was employed to generate three mutant LIM2 proteins with conversions of the second conserved cysteine to histidine (C2H), the fifth conserved cysteine to histidine (C5H), and the last conserved cysteine to aspartate (C8D). Metal coordination by the mutant proteins was evaluated by atomic absorption spectroscopy, Co(II) electronic spectroscopy, and 113Cd NMR spectroscopy. The results permit discrimination between various models of metal ion binding and suggest that the LIM domain is comprised of a S3N1 site generated from the four N-terminal candidate ligands (CX2CX17HX2C) and a S4 site generated from the four C-terminal candidate ligands (CX2CX17CX2C).