Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and ...cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25–100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase “rolling circle” activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential (“hyperbranched”) RCA amplification made possible by use of a second primer; the development of the “padlock” process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.
Understanding the diversity of dynamic structures and functions of DNA and RNA in biology requires tools that can selectively and intimately probe these biomolecules. Synthetic fluorescent ...nucleobases that can be incorporated into nucleic acids alongside their natural counterparts have emerged as a powerful class of molecular reporters of location and environment. They are enabling new basic insights into DNA and RNA, and are facilitating a broad range of new technologies with chemical, biological and biomedical applications. In this Review, we will present a brief history of the development of fluorescent nucleobases and explore their utility as tools for addressing questions in biophysics, biochemistry and biology of nucleic acids. We provide chemical insights into the two main classes of these compounds: canonical and non-canonical nucleobases. A point-by-point discussion of the advantages and disadvantages of both types of fluorescent nucleobases is made, along with a perspective into the future challenges and outlook for this burgeoning field.
The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer ...chemistry, biomaterials and hydrogels, dynamic combinatorial chemistry, organic synthesis, and chemical biology. Here we outline chemical developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomolecules are described. The basic chemical properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction’s mechanism and how reactant structure controls rates and equilibria in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified especially fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addition, the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A number of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomolecules and biomaterials.
The formation of oximes and hydrazones is widely used in chemistry and biology as a molecular conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow ...rate of reaction has hindered its utility. Here, we report that simple, commercially available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst–imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concentrations of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1–2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the ortho-carboxylate group in the anthranilate catalysts serves to aid in intramolecular proton transfer during imine and hydrazone formation.
A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified ...bases have been identified, research into the world of RNA modification - the epitranscriptome - is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.
Methods for RNA functionalization at specific sites are in high demand but remain a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have ...described acylimidazole reagents that react in high yields at 2′-OH groups stochastically at nonbase-paired regions, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here, we describe a DNA-directed strategy for in vitro functionalization of RNA at site-localized 2′-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with an acylimidazole reagent is then carried out, providing high yields of 2′-OH conjugation at predetermined sites. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach are carried out to control localized ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple and novel strategy for site-selective labeling and control of RNAs, potentially of any length and origin.
Glycation, the term for non-enzymatic covalent reactions between aldehyde metabolites and nucleophiles on biopolymers, results in deleterious cellular damage and diseases. Since ...Parkinsonism-associated protein DJ-1 was proposed as a novel deglycase that directly repairs glycated adducts, it has been considered a major contributor to glycation damage repair. Recently, an interesting debate over the mechanism of glycation repair by DJ-1 has emerged, focusing on whether the substrate of DJ-1 is glycated adducts or the free small aldehydes. The physiological significance of DJ-1 on glycation defense also remains in question. This debate is complicated by the fact that glycated biomolecular adducts are in rapid equilibrium with free aldehydes. Here, we summarize experimental evidence for the two possibilities, highlighting both consistencies and conflicts. We discuss the experimental complexities from a mechanistic perspective, and suggest classes of experiments that should help clarify this debate.
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Debate over repair of glycation by the enzyme DJ-1 has emerged, centering on whether the substrate is glycated biomacromolecules (deglycase activity) or free small-molecule aldehydes (glyoxalase activity). Jun and Kool summarize experimental evidence for each possibility, highlighting both consistencies and conflicts, and discuss the experimental complexities from a mechanistic perspective.
RNA plays pivotal roles in most cellular processes, serving as both the traditional carrier of genetic information and as a key regulator of cellular functions. The advent of chemical technologies ...has contributed critically to the analysis of cellular RNA structures, functions, and interactions. Many of these methods and molecules involve the utilization of chemically reactive handles in RNAs, either introduced externally or inherent within the polymer itself. Among these handles, the 2′-hydroxyl (2′-OH) group has emerged as an exceptionally well-suited and general chemical moiety for the modification and profiling of RNAs in intracellular studies. In this review, we provide an overview of the recent advancements in intracellular applications of acylation at the 2′-OH group of RNA. We outline progress made in probing RNA structure and interactomes, controlling RNA function, RNA imaging, and analyzing RNA-small molecule interactions, all achieved in living cells through this simple chemical handle on the biopolymer.
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The use of reagents that acylate 2′-OH groups in RNA has traditionally been employed for structure mapping, mainly in vitro. Xiao et al. outline recent rapid growth of chemical methods that have been developed for intracellular applications, including mapping in-cell RNA structures/interactions, caging RNA, conjugating labels, and profiling small-molecule interactions.
Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow ...for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)
and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.
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