Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer.

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.

Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles.

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5΄-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation.

Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences.

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K(+) solution, whereas a potentially photoreactive basket conformation is favored in Na(+) solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na(+) solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K(+) solution. In the current study, Na(+) was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K(+) solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K(+) solution are dynamic and able to access photoreactive conformations more easily than in Na(+) solution.

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle.

Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=(m)CG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=(m)CG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis.

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis-syn thymidine-thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.

Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Preparation of site-specific T=mCG cis-syn cyclobutane dimer-containing template and its error-free bypass by yeast and human polymerase η.

C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine dimer (CPD) formation. In response, cells have evolved DNA damage bypass polymerases, of which polymerase η (pol η) appears to be specifically adapted to synthesize past cis-syn CPDs. Although T=T CPDs are stable, CPDs containing C or 5-methylcytosine ((m)C) are not and spontaneously deaminate to U or T at pH 7 and 37 °C over a period of hours or days, making their preparation and study difficult. Furthermore, there is evidence to suggest that, depending on solvent polarity, a C or an (m)C in a CPD can adopt three tautomeric forms, one of which could code as T. Although many in vitro studies have established that synthesis past T or U in a CPD by pol η occurs in a highly error-free manner, the only in vitro evidence that synthesis past C or (m)C in a CPD also occurs in an error-free manner is for an (m)C in the 5'-position of an (m)C=T CPD. Herein, we describe the preparation and characterization of an oligodeoxynucleotide containing a CPD of a T(m)CG site, one of the major sites of C methylation and C-to-T mutations found in the p53 gene of basal and squamous cell cancers. We also demonstrate that both yeast and human pol η synthesize past the 3'-(m)C CPD in a >99% error-free manner, consistent with the highly water-exposed nature of the active site.

Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.

Roles of free radicals in type 1 phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates.

Detailed analyses of the electron spin resonance (ESR) spectra, cell viability, and DNA degradation studies are presented for the photolyzed Type I phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates. The ESR studies provided evidence that copious free radicals can be generated from these N-H, N-S, and S-O containing compounds upon photoirradiation with UV/visible light. The analyses of spectral data allowed us to identify the free radical species. The cell viability studies showed that these agents after exposure to light exert cytotoxicity to kill cancer cells (U937 leukemia cell lines HTC11, KB, and HT29 cell lines) in a dosage- and time-dependent manner. We examined a possible pathway of cell death via DNA degradation by a plasmid cleavage assay for several compounds. The effects of photosensitization with benzophenone in the presence of oxygen were examined. The studies indicate that planar tricyclic amines and sulfenamides tend to form π-electron delocalized aminyl radicals, whereas nonplanar ones tend to yield nitroxide radicals resulting from the recombination of aminyl radicals with oxygen. The ESR studies coupled with the results of cell viability measurements and DNA degradation reveal that planar N-centered radicals can provide higher potency in cell death and allow us to provide some insights on the reaction mechanisms. We also found the formation of azatropylium cations possessing high aromaticity derived from azepines can facilitate secondary electron transfer to form toxic O2(•-) radicals, which can further exert oxidative stress and cause cell death.

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†.

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.

Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate.

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase η opposite the T that results from deamination of the methylC ((m)C) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of (m)C deamination in a CPD has not been previously studied. We now report that deamination of a T(m)C CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a T(m)C CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Because the (m)Cs undergoing deamination are in similar steric environments, the difference in rate appears to be a consequence of a difference in the flexibility and compression of the two sites due to DNA bending. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a T(m)CG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced (m)C to T mutation than one positioned against the surface. These results indicate that rotational position may play an important role in the formation of UV-induced C to T mutation hotspots, as well as in the mutagenic mechanism of other DNA lesions.

Inhibition of heat shock transcription factor binding by a linear polyamide binding in an unusual 1:1 mode.

Heat shock proteins (HSPs) are known to protect cells from heat, oxidative stress, and the cytotoxic effects of drugs, and thus can enhance cancer cell survival. As a result, HSPs are a newly emerging class of protein targets for chemotherapy. Among the various HSPs, the HSP70 family is the most highly conserved and prevalent. Herein we describe the development of a ß-alanine rich linear polyamide that binds the GGA heat shock elements (HSEs) 3 and 4 in the HSP70 promoter in an unusual 1:1 mode and inhibits heat shock transcription factor 1 (HSF1) binding in vitro.

Native mRNA antisense-accessible sites library for the selection of antisense oligonucleotides, PNAs, and siRNAs.

A procedure for rapidly generating a library of antisense-accessible sites on native mRNAs (mRNA antisense-accessible sites library [MASL]) is described that involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific polymerase chain reaction (PCR). Antisense phosphorothioate oligodeoxynucleotides (ODNs), peptide nucleic acids (PNAs), and small interfering RNAs (siRNAs) can then be identified by screening against the antisense-accessible sites. The utility of this methodology is demonstrated for the identification of more effective inhibitors of inducible nitric oxide synthase (iNOS) induction than have previously been reported. This method may also be useful for constraining folding calculations of native mRNAs and for designing mRNA imaging probes.

Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury.

In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.

Methyl CpG binding protein 2 (MeCP2) enhances photodimer formation at methyl-CpG sites but suppresses dimer deamination.

Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of C→T mutations, but occurs with a half life of ∼50 000 years. In contrast, cytosine within sunlight induced cyclobutane dipyrimidine dimers (CPD's), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlate with C→T mutation hotspots in skin cancers. MeCP2 binds to mCG sites and acts as a transcriptional regulator and chromatin modifier affecting thousands of genes, but its effect on CPD formation and deamination is unknown. We report that the methyl CpG binding domain of MeCP2 (MBD) greatly enhances C=mC CPD formation at a TCmCG site in duplex DNA and binds with equal or better affinity to the CPD-containing duplex compared with the undamaged duplex. In comparison, MBD does not enhance T=mC CPD formation at a TTmCG site, but instead increases CPD formation at the adjacent TT site. MBD was also found to completely suppress deamination of the T=mCG CPD, suggesting that MeCP2 may have the capability to both suppress UV mutagenesis at PymCpG sites as well as enhance it.

Photocrosslinking of human telomeric G-quadruplex loops by anti cyclobutane thymine dimer formation.

The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)(3)], forms a basket structure in the presence of Na(+) and may form multiple equilibrating structures in the presence of K(+) with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)(3)] in the presence of Na(+) results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K(+), however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na(+). Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.

Ability of the Putative Decomposition Products of 2,3-dioxetanes of Indoles to Photosensitize Cyclobutane Pyrimidine Dimer (CPD) Formation and its Implications for the "Dark" (Chemisensitized) Pathway to CPDs in Melanocytes†.

The formation of cyclobutane pyrimidine dimers (CPDs) by a "dark" pathway in melanocytes has been attributed to chemisensitization by dioxetanes produced from peroxynitrite oxidation of melanin or melanin precursors. These dioxetanes are proposed to decompose to triplet state compounds which sensitize CPD formation by triplet-triplet energy transfer. To determine whether such compounds are capable of sensitizing CPD formation, the putative decomposition products of 2,3-dioxetanes of variously substituted indoles were synthesized and their triplet state energies determined at 77 K. Their ability to photosensitize CPD formation was determined by an enzyme-coupled gel electrophoresis assay in comparison with norfloxacin (NFX) which has the lowest triplet energy known to sensitize CPD formation. The decomposition products of 2,3-dioxetanes of 5-hydroxy and 5,6-dimethoxy indoles used as models for melanin precursors had lower triplet energies and were incapable of photosensitizing CPD formation. Theoretical calculations suggest that the decomposition products of the 2,3-dioxetanes of melanin precursors DHI and DHICA will have similarly low triplet energies. Decomposition products of the 2,3-dioxetanes of indoles lacking oxygen substituents had higher triplet energies than NFX and were capable of photosensitizing CPD formation, suggesting that peroxynitrite oxidation of tryptophan could play a hitherto unrecognized role in the dark pathway to CPDs.

Biotinylated quercetin as an intrinsic photoaffinity proteomics probe for the identification of quercetin target proteins.

Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC-MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC-MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins.