Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Adam P. Silverman is active.

Publication


Featured researches published by Adam P. Silverman.


Journal of Molecular Biology | 2009

Engineered Cystine-Knot Peptides that Bind αvβ3 Integrin with Antibody-Like Affinities

Adam P. Silverman; Aron M. Levin; Jennifer L. Lahti; Jennifer R. Cochran

The alpha(v)beta(3) integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to alpha(v)beta(3) integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized alpha(v)beta(3) integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing alpha(v)beta(3) integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to alpha(v)beta(3) integrins and had only minimal or no binding to alpha(v)beta(5), alpha(5)beta(1), and alpha(iib)beta(3) integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for alpha(v)beta(3) and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against alpha(v)beta(3) integrins resulted in peptides that retained high affinities for alpha(v)beta(3) and had increased specificities for alpha(v)beta(3) over alpha(iib)beta(3) integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.


Nucleic Acids Research | 2005

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Adam P. Silverman; Eric T. Kool

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.


Proceedings of the National Academy of Sciences of the United States of America | 2011

Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and αvβ3 integrin

Niv Papo; Adam P. Silverman; Jennifer L. Lahti; Jennifer R. Cochran

Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and αvβ3 integrin. Thus, agents that inhibit both receptors would have important therapeutic potential. Here, we used an antagonistic VEGF ligand as a molecular scaffold to engineer dual-specific proteins that bound to VEGFR2 and αvβ3 integrin with antibody-like affinities and inhibited angiogenic processes in vitro and in vivo. Mutations were introduced into a single-chain VEGF (scVEGF) ligand that retained VEGFR2 binding, but prevented receptor dimerization and activation. Yeast-displayed scVEGF mutant libraries were created and screened by high-throughput flow cytometric sorting to identify several variants that bound with high affinity to both VEGFR2 and αvβ3 integrin. These engineered scVEGF mutants were specific for αvβ3 integrin and did not bind to the related integrins αvβ5, αiibβ3, or α5β1. In addition, surface plasmon resonance and cell binding assays showed that dual-specific scVEGF proteins can simultaneously engage both receptors. Compared to monospecific scVEGF mutants that bind VEGFR2 or αvβ3 integrin, dual-specific scVEGF proteins more strongly inhibited VEGF-mediated receptor phosphorylation, capillary tube formation, and proliferation of endothelial cells cultured on Matrigel or vitronectin-coated surfaces. Moreover, dual specificity conferred strong inhibition of VEGF-mediated blood vessel formation in Matrigel plugs in vivo, whereas monospecific scVEGF mutants that bind VEGFR2 or αvβ3 integrin were only marginally effective. Instead of relying on antibody associating domains or physical linkage, this work highlights an approach to creating dual-specific proteins where additional functionality is introduced into a protein ligand to complement its existing biological properties.


Advances in Clinical Chemistry | 2007

Oligonucleotide Probes for RNA‐Targeted Fluorescence In Situ Hybridization

Adam P. Silverman; Eric T. Kool

The need for accurate and rapid methodology for detecting cells in environmental and clinical samples has led to the development of in situ detection methods, where fixed or intact cells can be imaged directly. In this chapter, we focus on the use of labeled oligonucleotide probes in fluorescence in situ hybridization (FISH). We give an overview of FISH probe design, covering issues of affinity and specificity of probes, probe backbone options, cellular targets, and accessibility of target sequences. Decisions that must be made to design optimal probes are evaluated, and available resources to assist in probe design, such as secondary structure, Tm calculation, and site accessibility software, are discussed. We cover different types of FISH probes that have been reported in the recent literature, including standard fluorescently labeled oligonucleotide probes and newer classes of quenched oligonucleotide probes: molecular beacons and quenched autoligation probes. Advantages and disadvantages of the different probe types are examined and recent literature applications are discussed. The current state of the art in the field as well as limitations and challenges in detection are evaluated.


Trends in Biotechnology | 2008

Developing therapeutic proteins by engineering ligand–receptor interactions

Douglas S. Jones; Adam P. Silverman; Jennifer R. Cochran

Ligand-receptor interactions govern myriad cell signaling pathways that regulate homeostasis and ensure that cells respond properly to stimuli. Growth factors, cytokines and other regulatory elements use these interactions to mediate cell responses, including proliferation, migration, angiogenesis, immune responses and cell death. Proteins that inhibit these processes have potential as therapeutics for cancer and autoimmune disorders, whereas proteins that stimulate these processes offer promise in regenerative medicine. Although much of the focus in this area over the past decade has been on monoclonal antibodies, recently there has been increased interest in the use of non-antibody proteins as therapeutic agents. Here, we review recent advances and accomplishments in the use of rational and combinatorial protein engineering approaches to developing ligands and receptors as agonists and antagonists against clinically important targets.


Journal of Molecular Recognition | 2011

Cystine-knot peptides engineered with specificities for αIIbβ3 or αIIbβ3 and αvβ3 integrins are potent inhibitors of platelet aggregation

Adam P. Silverman; Mihalis Kariolis; Jennifer R. Cochran

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ∼20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin αIIbβ3, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (KD) constants for αIIbβ3 integrin ranging from 60 to 90 nM, and did not bind to αvβ3, αvβ5, or α5β1 integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both αIIbβ3 and αvβ3 integrins with KD values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to αvβ5 or α5β1 integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright


ChemBioChem | 2006

RNA-Templated Chemistry in Cells: Discrimination of Escherichia, Shigella and Salmonella Bacterial Strains With a New Two-Color FRET Strategy

Adam P. Silverman; Ellen Jo Baron; Eric T. Kool

Highly specific detection and identification of many microorganisms in the clinic remains a costly, timeand expertise-intensive process, despite the advent of molecular in situ probing technologies such as fluorescence in situ hybridization (FISH). Commercial FISH probes are now available for distinguishing broad differences in sequences of ribosomal RNAs, but they commonly fail to detect smaller variations that can be medically significant. An important example is the discrimination of genetically closely-related Escherichia coli, Salmonella, and Shigella species. Salmonella and Shigella are agents of food-borne and travelers’ diarrhea, and can occasionally invade the intestinal mucosa to cause systemic disease. Some strains or clones of E. coli are pathogenic in the gastrointestinal tract by virtue of producing cytotoxins, enterotoxins, or by adherence mechanisms, but other strains of E. coli are harmless gastrointestinal colonizers. The typical methods for laboratory diagnosis of bacterial diarrhea caused by these organisms is to inoculate fecal material onto a series of agar plates made with selective or differential components and examine the resulting bacterial colonies after overnight incubation. Colony morphologies typical of the different pathogens are subcultured and characterized further using biochemical and serological methods. A number of new methods for bacterial detection have appeared in the literature in recent years; however, most fail to offer much utility for the clinical setting. One other RNA ACHTUNGTRENNUNGdetection methodology, molecular beacons, has been used to image human cells and in one case, fixed bacterial cells, but no reports exist on their use in live bacteria. QUAL probes are the only detection method thus far reported to successfully differentiate live bacteria. In order for a detection scheme to be routinely useful in a clinical laboratory, several requirements should ideally be met: 1) the assay must be relatively low cost, simple, and employ equipment that a typical lab already owns (for example, fluorescence microscope with a simple filter set) ; 2) the results should be easy and unambiguous to read; 3) results should be rapidly obtained in-house; and of course, 4) the results should have adequate sensitivity (preferably >95%) and specificity for the patient types and samples being tested (sensitivity is the probability of a positive test among patients with the infection; specificity is the probability of a negative test among patients without the infection). Quenched autoligation (QUAL) probes have recently been introduced as a new molecular strategy for in situ RNA detection and imaging. QUAL probes are highly selective oligonucleotide probes that target DNA or RNA in solution or RNA in cells. The probes function in pairs: one nucleophilic probe with a 3’-terminal phosphorothioate group, and an electrophilic probe containing an internal fluorescein (FAM) moiety and a 5’-“dabsylate” quencher/leaving group. When the probes bind a target adjacent to one another, the quencher is displaced, leading to a fluorescence signal. Sequence discrimination is achieved by positioning a mismatch site at the center of one of the probes. Furthermore, probes can undergo turnover (and hence, isothermal amplification of the signal) by use of a linker attached to the dabsylate group that destabilizes the ligated product complex, promoting dissociation from the RNA. In clinical settings it would be advantageous if small genetic differences in cells could be discriminated by different colors under the laboratory microscope, tagging multiple probes with unique colors. A previous approach taken to expand the range of colors with QUAL probes was to simply replace an internal fluorescein with a different fluorophore. However, in practice this approach is problematic for several reasons. First, very few fluorophores are available as phosphoramidite derivatives for facile internal coupling to a DNA sequence, so conjugation to DNA requires time-consuming and often difficult post-synthetic coupling steps that can be incompatible with the labile dabsylate group. Second, incomplete quenching of red-shifted fluorophores by dabsylate lowers the signal-tonoise ratio, making it unlikely that good discrimination in bacteria would be achieved using other colors. Third, different dyes must be excited by different wavelengths of light, requiring multiple filter sets for imaging, followed by digital overlaying of false-color images. This can make it difficult to make a direct judgment of which color is dominant. In the present work, we describe a new method for multicolor rRNA-targeted in situ imaging that overcomes these problems, allowing a ACHTUNGTRENNUNGsimultaneous visual readout of two colors, and we test the ACHTUNGTRENNUNGapproach in discrimination of pathogenic strains of bacteria. The goal of our new probe design was to use two colors that could be imaged under a single filter set (note, however, that the design offers the possibility of using additional FRET pairs to distinguish three or more sequences). The strategy involves the use of a single dye (FAM) to generate a green signal, and a FRET dye pair (FAM+TAMRA) to generate a red signal using the same excitation as with the FAM alone (Scheme 1). Using this approach we designed specific probes for 16S ribosomal RNA site 821–843, which has a single nucleotide difference between the closely related E. coli and Salmonella enterica. These bacteria are highly homologous and differ [a] A. P. Silverman, E. T. Kool Department of Chemistry, Stanford University Stanford, CA 94305-5080 (USA) Fax: (+1)650-725-0295 E-mail : [email protected] [b] E. J. Baron Department of Pathology, Stanford University Medical Center Stanford, CA 94035-5629 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.


Chemistry: A European Journal | 2008

Steric Effects in RNA Interference: Probing the Influence of Nucleobase Size and Shape

Alvaro Somoza; Adam P. Silverman; Rand M. Miller; Jijumon Chelliserrykattil; Eric T. Kool

Nonpolar nucleosides with varying size and shape have been used to study the hydrogen-bonding stabilization and steric effects on RNA interference. The uracil and adenine residues of siRNA guide strands have been replaced by nonpolar isosteres of uracil and adenine and by steric variants. RNAi experiments targeting Renilla luciferase mRNA have shown close correlation between siRNA thermal stability and gene suppression. Interestingly, siRNA modified at position 7 on the guide strand does not follow this correlation, having substantial RNAi activity despite low thermal stability. Sequence-selectivity studies were carried out at this position with mutated target mRNAs and nucleobase analogues with varied size (2,4-difluoro- and 2,4-dichlorobenzene) and different shape (2,3-dichlorobenzene, 4-methylbenzimidazole). The results point out the importance of nucleobase shape and steric effects in RNA interference.


PLOS Computational Biology | 2009

Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein

Jennifer L. Lahti; Adam P. Silverman; Jennifer R. Cochran

Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.


Biochemistry | 2008

Probing the Active Site Steric Flexibility of HIV-1 Reverse Transcriptase: Different Constraints for DNA- versus RNA-Templated Synthesis

Adam P. Silverman; Scott J. Garforth; Vinayaka R. Prasad; Eric T. Kool

The steric flexibility or rigidity of polymerase active sites may play an important role in their fidelity of nucleic acid synthesis. In this regard, reverse transcriptases offer an unusual opportunity to compare two enzymatic activities that proceed in the same active site. For HIV-1 reverse transcriptase, reverse transcription (RNA-templated synthesis) is known to proceed with lower fidelity than DNA-templated synthesis. Here, we describe the use of a set of variably sized nonpolar thymidine and uracil mimics as molecular rulers to probe the active site steric constraints of HIV-1 RT, and for the first time, we directly compare the functional flexibility of these two activities. Steady-state kinetics of incorporation for natural dNTPs opposite unnatural template bases as well as for unnatural dNTPs opposite natural template bases are reported for the DNA-templated DNA synthesis, and comparison is made with recent data for the RNA-templated activity. Kinetics for extension beyond a base pair containing the analogue template bases are also reported both for RNA and DNA templates. Our results show that the DNA-dependent polymerization by HIV-RT is highly sensitive to size, strongly biasing against both too-small and too-large base pairs, while, by contrast, the RNA-dependent polymerization is only biased against analogues that are too small, and is much more accepting of larger base pairs. In addition, base pair extension with HIV-RT is found to be relatively insensitive to varied base pair size, consistent with its high mutagenicity. Overall, the data show greater rigidity with a DNA template as compared with an RNA template, which correlates directly with the higher fidelity of the DNA-templated synthesis. Possible structural explanations for these differences are discussed. We also report kinetics data for two HIV-1 RT mutants reported to have altered fidelity (F61A and K65R) using DNA templates containing nonpolar base analogues, and find that one of these (F61A) is a high-fidelity enzyme that appears to be sensitive to a loss of hydrogen-bonding groups.

Collaboration


Dive into the Adam P. Silverman's collaboration.

Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Niv Papo

Weizmann Institute of Science

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Douglas A. Jones

Massachusetts Institute of Technology

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Top Co-Authors

Avatar
Researchain Logo
Decentralizing Knowledge