Crystal structures of sulfonamide protected bicyclic guanidines: (S)-8-{[(tert-butyl-dimethyl-sil-yl)-oxy]meth-yl}-1-[(2,2,4,6,7-penta-methyl-2,3-di-hydro-benzo-furan-5-yl)sulfon-yl]-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium tri-fluoro-methane-sulfonate and (S)-8-(iodo-meth-yl)-1-tosyl-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium iodide.

Two compounds, (S)-8-{[(tert-butyl-dimethyl-sil-yl)-oxy]meth-yl}-1-[(2,2,4,6,7-penta-methyl-2,3-di-hydro-benzo-furan-5-yl)sulfon-yl]-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium tri-fluoro-methane-sulfonate, C27H46N3O4SSi+·CF3O3S-, (1) and (S)-8-(iodo-meth-yl)-1-tosyl-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium iodide, C15H21IN3O2S+·I-, (2), have been synthesized and characterized. They are bicyclic guanidinium salts and were synthesized from N-(tert-but-oxy-carbon-yl)-l-me-thio-nine (Boc-l-Met-OH). The guanidine is protected by a 2,2,4,6,7-penta-methyl-dihydro-benzo-furan-5-sulfonyl (Pbf, 1) or a tosyl (2) group. In the crystals of both compounds, the guanidinium group is almost planar and the N-H forms an intra-molecular hydrogen bond in a six-membered ring to the oxygen atom of the sulfonamide protecting group.

Identification and detection of microRNA kidney disease biomarkers in liquid biopsies.

PURPOSE OF REVIEW: MicroRNAs (miRNAs) are emerging rapidly as a novel class of biomarkers of major organ disorders, including kidney diseases. However, current PCR-based detection methods are not amenable to development for high-throughput, cost-effective miRNA biomarker quantification. RECENT FINDINGS: MiRNA biomarkers show significant promise for diagnosis and prognosis of kidney diseases, including diabetic kidney disease, acute kidney injury, IgA nephropathy and delayed graft function following kidney transplantation. A variety of novel methods to detect miRNAs in liquid biopsies including urine, plasma and serum are being developed. As miRNAs are functional transcripts that regulate the expression of many protein coding genes, differences in miRNA profiles in disease also offer clues to underlying disease mechanisms. SUMMARY: Recent findings highlight the potential of miRNAs as biomarkers to detect and predict progression of kidney diseases. Developing in parallel, novel methods for miRNA detection will facilitate the integration of these biomarkers into rapid routine clinical testing and existing care pathways. Validated kidney disease biomarkers also hold promise to identify novel therapeutic tools and targets. VIDEO ABSTRACT: http://links.lww.com/CONH/A43.

The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4+ memory T cells.

Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular ß-chain variable (TRBV) regions of αß T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.

The Contribution of Germline Pathogenic Variants in Breast Cancer Genes to Contralateral Breast Cancer Risk in BRCA1/BRCA2/PALB2-Negative Women.

BACKGROUND: Contralateral breast cancer (CBC) is associated with younger age at first diagnosis, family history and pathogenic germline variants (PGVs) in genes such as BRCA1, BRCA2 and PALB2. However, data regarding genetic factors predisposing to CBC among younger women who are BRCA1/2/PALB2-negative remain limited. METHODS: In this nested case-control study, participants negative for BRCA1/2/PALB2 PGVs were selected from the WECARE Study. The burden of PGVs in established breast cancer risk genes was compared in 357 cases with CBC and 366 matched controls with unilateral breast cancer (UBC). The samples were sequenced in two phases. Whole exome sequencing was used in Group 1, 162 CBC and 172 UBC (mean age at diagnosis: 42 years). A targeted panel of genes was used in Group 2, 195 CBC and 194 UBC (mean age at diagnosis: 50 years). Comparisons of PGVs burdens between CBC and UBC were made in these groups, and additional stratified sub-analysis was performed within each group according to the age at diagnosis and the time from first breast cancer (BC). RESULTS: The PGVs burden in Group 1 was significantly higher in CBC than in UBC (p = 0.002, OR = 2.5, 95CI: 1.2-5.6), driven mainly by variants in CHEK2 and ATM. The proportions of PGVs carriers in CBC and UBC in this group were 14.8% and 5.8%, respectively. There was no significant difference in PGVs burden between CBC and UBC in Group 2 (p = 0.4, OR = 1.4, 95CI: 0.7-2.8), with proportions of carriers being 8.7% and 8.2%, respectively. There was a significant association of PGVs in CBC with younger age. Metanalysis combining both groups confirmed the significant association between the burden of PGVs and the risk of CBC (p = 0.006) with the significance driven by the younger cases (Group 1). CONCLUSION: In younger BRCA1/BRCA2/PALB2-negative women, the aggregated burden of PGVs in breast cancer risk genes was associated with the increased risk of CBC and was inversely proportional to the age at onset.

Explorations into Peptide Nucleic Acid Contrast Agents as Emerging Scaffolds for Breakthrough Solutions in Medical Imaging and Diagnosis.

Peptide nucleic acids (PNAs, nucleic acid analogues with a peptide backbone rather than a phosphoribosyl backbone) have emerged as promising chemical agents in antigene or antisense therapeutics, as splicing modulators or in gene editing. Their main benefits, compared to DNA or RNA agents, are their biochemical stability and the lack of negative charges throughout the backbone, leading to negligible electrostatic interaction with the strand with which they are hybridizing. As a result, hybridization of PNA strands with DNA or RNA strands leads to higher binding energies and melting temperatures. A lack of natural transporters, however, necessitates the formation of PNA-containing chimeras or the formulation of nanoparticular cell delivery methods. Here, we set out to explore the progress made in using imaging agents based on PNAs in diagnostic applications and highlight selected developments and challenges.

Synthetic Peptides with Inadvertent Chemical Modifications Can Activate Potentially Autoreactive T Cells.

The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161-170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.

Detection of urinary microRNA biomarkers using diazo sulfonamide-modified screen printed carbon electrodes.

This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA (miRNA) quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects. Aberrant miRNA expression has been observed in several major human disorders, and we have identified a urinary miRNA signature for DKD. MiRNAs therefore have considerable promise as disease biomarkers, and techniques to quantify these transcripts from clinical samples have significant clinical and commercial potential. Current RT-qPCR-based methods require technical expertise, and more straightforward methods such as electrochemical detection offer attractive alternatives. We describe a method to detect urinary miRNAs using diazo sulfonamide-modified screen printed carbon electrode-based biosensors that is amenable to parallel analysis. These sensors showed a linear response to buffered miR-21, with a 17 fM limit of detection, and successfully discriminated between urine samples (n = 6) from DKD patients and unaffected control subjects (n = 6) by differential miR-192 detection. Our technique for quantitative miRNA detection in liquid biopsies has potential for development as a platform for non-invasive high-throughput screening and/or to complement existing diagnostic procedures in disorders such as DKD.

Cancer Risks Associated With Germline PALB2 Pathogenic Variants: An International Study of 524 Families.

PURPOSE: To estimate age-specific relative and absolute cancer risks of breast cancer and to estimate risks of ovarian, pancreatic, male breast, prostate, and colorectal cancers associated with germline PALB2 pathogenic variants (PVs) because these risks have not been extensively characterized. METHODS: We analyzed data from 524 families with PALB2 PVs from 21 countries. Complex segregation analysis was used to estimate relative risks (RRs; relative to country-specific population incidences) and absolute risks of cancers. The models allowed for residual familial aggregation of breast and ovarian cancer and were adjusted for the family-specific ascertainment schemes. RESULTS: We found associations between PALB2 PVs and risk of female breast cancer (RR, 7.18; 95% CI, 5.82 to 8.85; P = 6.5 × 10-76), ovarian cancer (RR, 2.91; 95% CI, 1.40 to 6.04; P = 4.1 × 10-3), pancreatic cancer (RR, 2.37; 95% CI, 1.24 to 4.50; P = 8.7 × 10-3), and male breast cancer (RR, 7.34; 95% CI, 1.28 to 42.18; P = 2.6 × 10-2). There was no evidence for increased risks of prostate or colorectal cancer. The breast cancer RRs declined with age (P for trend = 2.0 × 10-3). After adjusting for family ascertainment, breast cancer risk estimates on the basis of multiple case families were similar to the estimates from families ascertained through population-based studies (P for difference = .41). On the basis of the combined data, the estimated risks to age 80 years were 53% (95% CI, 44% to 63%) for female breast cancer, 5% (95% CI, 2% to 10%) for ovarian cancer, 2%-3% (95% CI females, 1% to 4%; 95% CI males, 2% to 5%) for pancreatic cancer, and 1% (95% CI, 0.2% to 5%) for male breast cancer. CONCLUSION: These results confirm PALB2 as a major breast cancer susceptibility gene and establish substantial associations between germline PALB2 PVs and ovarian, pancreatic, and male breast cancers. These findings will facilitate incorporation of PALB2 into risk prediction models and optimize the clinical cancer risk management of PALB2 PV carriers.

Germline pathogenic variants in PALB2 and other cancer-predisposing genes in families with hereditary diffuse gastric cancer without CDH1 mutation: a whole-exome sequencing study.

BACKGROUND: Germline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants. METHODS: We did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer. FINDINGS: Protein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant. INTERPRETATION: The results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families. FUNDING: UK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007-13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK.

Electrochemical detection of urinary microRNAs via sulfonamide-bound antisense hybridisation.

Altered serum and plasma microRNA (miRNA) expression profiles have been observed in numerous human diseases, with a number of studies describing circulating miRNA biomarkers for cancer diagnosis, prognosis and response to treatment, and recruitment to clinical trials for miRNA-based drug therapy already underway. Electrochemical detection of biomarkers in urine has several significant advantages over circulating biomarker analysis including safety, cost, speed and ease of conversion to the point of care environment. Consequently, much current research is underway to identify urinary miRNA biomarkers for a variety of pathologies including prostate and bladder malignancies, and renal disorders. We describe here a robust method capable of electrochemical detection of human urinary miRNAs at femtomolar concentrations using a complementary DNA-modified glassy carbon electrode. A miR-21-specific DNA hybridisation probe was immobilised onto a glassy carbon electrode modified by sulfonic acid deposition and subsequent chlorination. In our pilot system, the presence of synthetic mature miR-21 oligonucleotides increased resistance at the probe surface to electron transfer from the ferricyanide/ferrocyanide electrolyte. Response was linear for 10 nM-10 fM miR-21, with a limit of detection of 20 fM, and detection discriminated between miR-21, three point-mutated miR-21 sequences, and miR-16. We then demonstrated similar sensitivity and reproducibility of miR-21 detection in urine samples from 5 human control subjects. Our protocol provides a platform for future high-throughput screening of miRNA biomarkers in liquid biopsies.

The Discovery and Validation of Biomarkers for the Diagnosis of Esophageal Squamous Dysplasia and Squamous Cell Carcinoma.

The 5-year survival rate of esophageal cancer is less than 10% in developing countries, where more than 90% of these cancers are esophageal squamous cell carcinomas (ESCC). Endoscopic screening is undertaken in high incidence areas. Biomarker analysis could reduce the subjectivity associated with histologic assessment of dysplasia and thus improve diagnostic accuracy. The aims of this study were therefore to identify biomarkers for esophageal squamous dysplasia and carcinoma. A publicly available dataset was used to identify genes with differential expression in ESCC compared with normal esophagus. Each gene was ranked by a support vector machine separation score. Expression profiles were examined, before validation by qPCR and IHC. We found that 800 genes were overexpressed in ESCC compared with normal esophagus (P < 10(-5)). Of the top 50 genes, 33 were expressed in ESCC epithelium and not in normal esophagus epithelium or stroma using the Protein Atlas website. These were taken to qPCR validation, and 20 genes were significantly overexpressed in ESCC compared with normal esophagus (P < 0.05). TNFAIP3 and CHN1 showed differential expression with IHC. TNFAIP3 expression increased gradually through normal esophagus, mild, moderate and severe dysplasia, and SCC (P < 0.0001). CHN1 staining was rarely present in the top third of normal esophagus epithelium and extended progressively towards the surface in mild, moderate, and severe dysplasia, and SCC (P < 0.0001). Two novel promising biomarkers for ESCC were identified, TNFAIP3 and CHN1. CHN1 and TNFAIP3 may improve diagnostic accuracy of screening methods for ESCC. Cancer Prev Res; 9(7); 558-66. ©2016 AACR.

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification.

We set out to clone Bax-specific CD8+ T-cells from peripheral blood samples of primary chronic lymphocytic leukemia patients. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-aa sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (>95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild type (wt) sequence to generate functionally active peptides. Computer modeling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the TCR. This modified product formed <1% of the original P603 crude peptide preparation and <0.05% of the original 23 peptide mixture used for T-cell stimulation. The data presented here, illustrates the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlights the exquisite capacity of TCR to discriminate between structurally similar peptide sequences. Furthermore this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses. This article is protected by copyright. All rights reserved.

The human hyaluronan synthase 2 (HAS2) gene and its natural antisense RNA exhibit coordinated expression in the renal proximal tubular epithelial cell.

Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1ß or TGF-ß1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1ß induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-ß1 stimulation. Inhibition of IL-1ß-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed up-regulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic double-stranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation.

A conserved stem loop motif in the 5'untranslated region regulates transforming growth factor-ß(1) translation.

Transforming growth factor-ß(1) (TGF-ß(1)) regulates cellular proliferation, differentiation, migration, and survival. The human TGF-ß(1) transcript is inherently poorly translated, and translational activation has been documented in relation to several stimuli. In this paper, we have sought to identify in cis regulatory elements within the TGF-ß(1) 5'Untranslated Region (5'UTR). In silico analysis predicted formation of stable secondary structure in a G/C-rich element between nucleotides +77 to +106, and demonstrated that this element is highly conserved across species. Circular dichroism spectroscopy confirmed the presence of secondary structure in this region. The proximal 5'UTR was inhibitory to translation in reporter gene experiments, and mutation of the secondary structure motif increased translational efficiency. Translational regulation of TGF-ß(1) mRNA is linked to altered binding of YB-1 protein to its 5'UTR. Immunoprecipitation-RT-qPCR demonstrated a high basal association of YB-1 with TGF-ß(1) mRNA. However, mutation of the secondary structure motif did not prevent interaction of YB-1 with the 5'UTR, suggesting that YB-1 binds to this region due to its G/C-rich composition, rather than a specific, sequence-dependent, binding site. These data identify a highly conserved element within the TGF-ß(1) 5'UTR that forms stable secondary structure, and is responsible for the inherent low translation efficiency of this cytokine.

Recognition and discrimination of DNA quadruplexes by acridine-peptide conjugates.

We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface plasmon resonance measurements revealed that all compounds bound to the human telomeric quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were used to model the effects of substituent orientation and peptide sequence. Modelling and experiment were in agreement that placement of the peptide over the face of the acridine is detrimental to binding affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that bound best displayed quadruplex affinities in the 1-5 nM range and at least 10-fold discrimination between the quadruplexes studied.

Surface plasmon resonance for probing quadruplex folding and interactions with proteins and small molecules.

Surface plasmon resonance is a technique for detecting binding events at the surface of a thin metal film. Through the commercial availability of instrumentation and sensor chips, the technique has found widespread application for determining the affinity and kinetics of macromolecular interactions. A variety of quadruplex forming oligonucleotides have been immobilized to sensor chips to permit analysis of their binding interactions with both small molecule and protein analytes. The fold of the quadruplex must be maintained through an appropriate choice of buffer, and care must be taken to ensure that data interpretation is not hampered by non-specific binding and adsorption of the analyte to the sensor surface and instrument. Affinity constants determined by surface plasmon resonance for interactions with quadruplexes correlate meaningfully with other methods, such as UV-visible and fluorescence titrations, enzyme linked immunosorbent assay, thermal melting studies and telomerase inhibition. Kinetic measurements of the association and dissociation of duplexes of quadruplex forming oligonucleotides and their complementary strands have enabled calculation of the folding and unfolding rates of the quadruplex itself, and determination of its stability as a function of buffer composition.

Discovery of G-quadruplex stabilizing ligands through direct ELISA of a one-bead-one-compound library.

We describe the identification of small-molecule G-quadruplex ligands using a direct ELISA screen of a one-bead-one-compound library of unnatural polyamides displayed on a branched linker with a biotin tag. This general purpose parallel screen for small molecule-oligonucleotide interactions was validated by surface plasmon resonance and ELISA of resynthesized compounds. Linear polyamides displayed similar rankings in their affinity for quadruplex as their branched counterparts. Quadruplex affinity as judged by these surface based techniques was a useful predictor of the ability of the ligands to stabilize the quadruplex to thermal unfolding in solution.

Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein.

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.