Epigenetic MRI: Noninvasive imaging of DNA methylation in the brain.

Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here, we present a method, epigenetic MRI (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene-expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C-labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.

DNAzyme-Catalyzed Site-Specific N-Acylation of DNA Oligonucleotide Nucleobases.

We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5'-carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.

Defining the substrate scope of DNAzyme catalysis for reductive amination with aliphatic amines.

Amines can be alkylated using various reactions, such as reductive amination of aldehydes. In this study, we sought DNAzymes as catalytic DNA sequences that promote reductive amination with aliphatic amines, including DNA-anchored peptide substrates with lysine residues. By in vitro selection starting with either N40 or N20 random DNA pools, we identified many DNAzymes that catalyze reductive amination between the DNA oligonucleotide-anchored aliphatic amino group of DNA-C3-NH2 (C3 = short three-carbon tether) and a DNA-anchored benzaldehyde group in the presence of NaCNBH3 as reducing agent. At pH 5.2, 6.0, 7.5, or 9.0 in the presence of various divalent metal ion cofactors including Mg2+, Mn2+, Zn2+ and Ni2+, the DNAzymes have kobs up to 0.12 h-1 and up to 130-fold rate enhancement relative to the DNA-splinted but uncatalyzed background reaction. However, analogous selection experiments did not lead to any DNAzymes that function with DNA-HEG-NH2 [HEG = long hexa(ethylene glycol) tether], or with short- and long-tethered DNA-AAAKAA and DNA-HEG-AAAKAA lysine-containing hexapeptide substrates (A = alanine, K = lysine). Including a variety of other amino acids in place of the neighboring alanines also did not lead to DNAzymes. These findings establish a practical limit on the substrate scope of DNAzyme catalysis for N-alkylation of aliphatic amines by reductive amination. The lack of DNAzymes for reductive amination with any substrate more structurally complex than DNA-C3-NH2 is likely related to the challenge in binding and spatially organizing those other substrates. Because other reactions such as aliphatic amine N-acylation are feasible for DNAzymes with DNA-anchored peptides, our findings show that the ability to identify DNAzymes depends strongly on both the investigated reaction and the composition of the substrate.

DNAzymes for amine and peptide lysine acylation.

DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However, finding DNAzymes for peptide lysine acylation is a substantial challenge. By using suitably reactive aryl ester acyl donors as the electrophiles, here we used in vitro selection to identify DNAzymes that acylate amines, including lysine side chains of DNA-anchored peptides. Some of the DNAzymes can transfer a small glutaryl group to an amino group. These results expand the scope of DNAzyme catalysis and suggest the future broader applicability of DNAzymes for sequence-selective lysine acylation of peptide and protein substrates.

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes.

The discovery of natural RNA enzymes (ribozymes) prompted the pursuit of artificial DNA enzymes (deoxyribozymes) by in vitro selection methods. A key motivation is the conceptual and practical advantages of DNA relative to proteins and RNA. Early studies focused on RNA-cleaving deoxyribozymes, and more recent experiments have expanded the breadth of catalytic DNA to many other reactions. Including modified nucleotides has the potential to widen the scope of DNA enzymes even further. Practical applications of deoxyribozymes include their use as sensors for metal ions and small molecules. Structural studies of deoxyribozymes are only now beginning; mechanistic experiments will surely follow. Following the first report 21 years ago, the field of deoxyribozymes has promise for both fundamental and applied advances in chemistry, biology, and other disciplines.

DNA-Catalyzed DNA Cleavage by a Radical Pathway with Well-Defined Products.

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N40 or N100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O2-• or HOO•) and hydrogen peroxide (H2O2) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H2O2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H2O2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn2+ and Mg2+, although including Mn2+ increases the activity, and Mn2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions.

DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.

T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.

DNA-Catalyzed Amide Hydrolysis.

DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.

Pursuing DNA catalysts for protein modification.

Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening have provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead surprisingly led to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 10(5)-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, we were able to identify deoxyribozymes for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion of chemically modified DNA nucleotides in the catalyst, which we have recently found to enable this cleavage reaction. In numerous other efforts, we have investigated DNA-catalyzed peptide side chain modification reactions. Key successes include nucleopeptide formation (attachment of oligonucleotides to peptide side chains) and phosphatase and kinase activities (removal and attachment of phosphoryl groups to side chains). Through all of these efforts, we have learned the importance of careful selection design, including the frequent need to develop specific "capture" reactions that enable the selection process to provide only those DNA sequences that have the desired catalytic functions. We have established strategies for identifying deoxyribozymes that accept discrete peptide and protein substrates, and we have obtained data to inform the key choice of random region length at the outset of selection experiments. Finally, we have demonstrated the viability of modular deoxyribozymes that include a small-molecule-binding aptamer domain, although the value of such modularity is found to be minimal, with implications for many selection endeavors. Advances such as those summarized in this Account reveal that DNA has considerable catalytic abilities for biochemically relevant reactions, specifically including covalent protein modifications. Moreover, DNA has substantially different, and in many ways better, characteristics than do small molecules or proteins for a catalyst that is obtained "from scratch" without demanding any existing information on catalyst structure or mechanism. Therefore, prospects are very strong for continued development and eventual practical applications of deoxyribozymes for peptide and protein modification.

Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions.

We evaluate the ability of hexahistidine (His6) tags on peptide and protein substrates to recruit deoxyribozymes for modifying those substrates. For two different deoxyribozymes, one that creates tyrosine-RNA nucleopeptides and another that phosphorylates tyrosine side chains, we find substantial improvements in yield, kobs, and Km for peptide substrates due to recruiting by His6/Cu(2+). However, the recruiting benefits of the histidine tag are not observed for larger protein substrates, likely because the tested deoxyribozymes either cannot access the target peptide segments or cannot function when these segments are presented in a structured protein context.

Catalytic DNA with phosphatase activity.

Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn(2+)-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase activity). The best deoxyribozyme decreases the half-life for phosphoserine hydrolysis from as high as >10(10) y to <1 h. The phosphatase activity also occurs with nonpeptidic substrates but with reduced efficiency, indicating a preference for phosphopeptides. The newly identified deoxyribozymes can function with multiple turnover using free peptide substrates, have activity in the presence of human cell lysate or BSA, and catalyze dephosphorylation of a larger protein substrate, suggesting broader application of DNA catalysts as artificial phosphatases.

DNA-Catalyzed Introduction of Azide at Tyrosine for Peptide Modification.

We show that DNA enzymes (deoxyribozymes) can introduce azide functional groups at tyrosine residues in peptide substrates. Using in vitro selection, we identified deoxyribozymes that transfer the 2'-azido-2'-deoxyadenosine 5'-monophosphoryl group (2'-Az-dAMP) from the analogous 5'-triphosphate (2'-Az-dATP) onto the tyrosine hydroxyl group of a peptide, which is either tethered to a DNA anchor or free. Some of the new deoxyribozymes are general with regard to the amino acid residues surrounding the tyrosine, while other DNA enzymes are sequence-selective. We use one of the new deoxyribozymes to modify free peptide substrates by attaching PEG moieties and fluorescent labels.

Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides.

Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.

Identification of Sequence-Selective Tyrosine Kinase Deoxyribozymes.

Deoxyribozymes (DNA enzymes) have been developed for a growing variety of chemical reactions, including with peptide substrates. We recently described the first tyrosine kinase deoxyribozymes, which lacked the ability to discriminate among peptide substrates on the basis of the amino acids surrounding the tyrosine residue. Those deoxyribozymes were identified by in vitro selection using a DNA-anchored peptide substrate in which the residues neighboring tyrosine were all alanine. Here, we performed in vitro selection for tyrosine kinase activity using three peptide substrates in which the neighboring residues included a variety of side chains. For one of these three peptides, we found numerous deoxyribozymes that discriminate strongly in favor of phosphorylating tyrosine when the surrounding residues are specifically those used in the selection process. Three different short peptide sequence motifs of 2-4 amino acids were required for catalysis by three unique deoxyribozymes. For a second peptide substrate, the selection process led to one deoxyribozyme which exhibits partial discrimination among peptide sequences. These findings establish the feasibility of identifying DNA enzymes that catalyze sequence-selective tyrosine phosphorylation, which suggests the downstream practical utility of such deoxyribozymes. More broadly, this outcome reinforces the conclusion that nucleic acid catalysts can discriminate among peptide substrates in the context of biochemically relevant reactions.

Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters.

We used in vitro selection to identify new DNA aptamers for two endocrine-disrupting compounds often found in treated and natural waters, 17ß-estradiol (E2) and 17α-ethynylestradiol (EE). We used equilibrium filtration to determine aptamer sensitivity/selectivity and dimethyl sulfate (DMS) probing to explore aptamer binding sites. The new E2 aptamers are at least 74-fold more sensitive for E2 than is a previously reported DNA aptamer, with dissociation constants (Kd values) of 0.6 µM. Similarly, the EE aptamers are highly sensitive for EE, with Kd of 0.5-1.0 µM. Selectivity values indicate that the E2 aptamers bind E2 and a structural analogue, estrone (E1), equally well and are up to 74-fold selective over EE. One EE aptamer is 53-fold more selective for EE over E2 or E1, but the other binds EE, E2, and E1 with similar affinity. The new aptamers do not lose sensitivity or selectivity in natural water from a local lake, despite the presence of natural organic matter (∼4 mg/L TOC). DMS probing suggests that E2 binding occurs in relatively flexible single-stranded DNA regions, an important finding for rational redesign of aptamers and their incorporation into sensing platforms. This is the first report of aptamers with strong selectivity for E2 and E1 over EE, or with strong selectivity for EE over E2 and E1. Such selectivity is important for achieving the goal of creating practically useful DNA-based sensors that can distinguish structurally similar estrogenic compounds in natural waters.

A generalizable DNA-catalyzed approach to peptide-nucleic acid conjugation.

We report DNA catalysts (deoxyribozymes) that join tyrosine-containing peptides to RNA and DNA in one step and without requiring protecting groups on either the peptide or the nucleic acid. Our previous efforts towards this goal required tethering the peptide to a DNA anchor oligonucleotide. Here, we established direct in vitro selection for deoxyribozymes that use untethered, free peptide substrates. This approach enables imposition of selection pressure via reduced peptide concentration and leads to preparatively useful lower apparent Km values of ∼100 µM peptide. Use of phosphorimidazolide (Imp) rather than triphosphate as the electrophile enables reactivity of either terminus (5' or 3') of both RNA and DNA. Our findings establish a generalizable means of joining unprotected peptide to nucleic acid in one step by using DNA catalysts identified by in vitro selection.

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA.

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.

DNA-catalyzed lysine side chain modification.

Catalyzing the covalent modification of aliphatic amino groups, such as the lysine (Lys) side chain, by nucleic acids has been challenging to achieve. Such catalysis will be valuable, for example, for the practical preparation of Lys-modified proteins. We previously reported the DNA-catalyzed modification of the tyrosine and serine hydroxy side chains, but Lys modification has been elusive. Herein, we show that increasing the reactivity of the electrophilic reaction partner by using 5'-phosphorimidazolide (5'-Imp) rather than 5'-triphosphate (5'-ppp) enables the DNA-catalyzed modification of Lys in a DNA-anchored peptide substrate. The DNA-catalyzed reaction of Lys with 5'-Imp is observed in an architecture in which the nucleophile and electrophile are not preorganized. In contrast, previous efforts showed that catalysis was not observed when Lys and 5'-ppp were used in a preorganized arrangement. Therefore, substrate reactivity is more important than preorganization in this context. These findings will assist ongoing efforts to identify DNA catalysts for reactions of protein substrates at lysine side chains.

DNA catalysts with tyrosine kinase activity.

We show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified deoxyribozymes that transfer the γ-phosphoryl group from a 5'-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools. Each deoxyribozyme requires Zn(2+), and most additionally require Mn(2+). The deoxyribozymes have little or no selectivity for the amino acid identities near the tyrosine, but they are highly selective for phosphorylating tyrosine rather than serine. Analogous GTP-dependent DNA catalysts were identified and found to have apparent Km(GTP) as low as ∼20 µM. These findings establish that DNA has the fundamental catalytic ability to phosphorylate the tyrosine side chain of a peptide substrate.

DNA catalysis of a normally disfavored RNA hydrolysis reaction.

We recently used in vitro selection to identify many deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5'-phosphate and 3'-hydroxyl termini. Alternatively, numerous deoxyribozymes have been identified for catalysis of RNA cleavage by 2'-hydroxyl transesterification, forming 2',3'-cyclic phosphate and 5'-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing deoxyribozyme leads either to transesterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis. An entirely new selection starting from a random DNA pool, using an all-RNA substrate and imposing the same selection pressure, also leads to RNA hydrolysis. Collectively, these results establish experimentally that small DNA sequences have the catalytic ability to direct a chemical reaction down a disfavored pathway, even when a more favorable mechanism is readily available. Our view of DNA catalysis is therefore expanded beyond merely increasing the rates of reactions that would have occurred more slowly without the catalyst.