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Cadmium (Cd) is one of the most toxic heavy metals. Exposure to Cd can impair the functions of the kidney, respiratory system, reproductive system and skeletal system. Cd2+-binding aptamers have been extensively utilized in the development of Cd2+-detecting devices; however, the underlying mechanisms remain elusive. This study reports four Cd2+-bound DNA aptamer structures, representing the only Cd2+-specific aptamer structures available to date. In all the structures, the Cd2+-binding loop (CBL-loop) adopts a compact, double-twisted conformation and the Cd2+ ion is mainly coordinated with the G9, C12 and G16 nucleotides. Moreover, T11 and A15 within the CBL-loop form one regular Watson–Crick pair and stabilize the conformation of G9. The conformation of G16 is stabilized by the G8–C18 pair of the stem. By folding and/or stabilizing the CBL-loop, the other four nucleotides of the CBL-loop also play important roles in Cd2+ binding. Similarly to the native sequence, crystal structures, circular dichroism spectrum and isothermal titration calorimetry analysis confirm that several variants of the aptamer can recognize Cd2+. This study not only reveals the underlying basis for the binding of Cd2+ ions with the aptamer, but also extends the sequence for the construction of novel metal–DNA complex.

The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation.

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2‐thiouridine using selenophosphate or geranyl‐pyrophosphate as cofactors. In this study, we explored thein vitrogeranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U₃₅was mutated to A₃₅(ASL‐tRNA^Lys_(s2U)UUto ASL‐tRNA^Ile_(s2U)AU). By examining the SelU cofactor geranyl‐pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl‐ and farnesyl‐pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end‐point analysis for SelU follows the order of ASL^Lys_(s2UUU)ASL^Gln_(s2UUG)>ASL^Glu_(s2UUC). These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU‐based antibiotics.

Owing to its great threat to human health and environment, Pb²⁺pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb²⁺-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb²⁺-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb²⁺is bound by the two G-tetrads in the center. Compared to K⁺-stabilized G-quadruplexes, the coordinating distance between Pb²⁺and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb²⁺-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb²⁺-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.

The syntheses of a series of novel 6‐aza‐2‐hydroxyimino‐5‐methylpyrimidine and related nucleosides are described. A suitably protected 2‐methylthiopyrimidine nucleoside was selected as the precursor for installing a hydroxyimino moiety at the C‐2 position. The starting nucleobase 6‐aza‐5‐methyl‐2‐thiouracil is prepared in two steps from thiosemicarbazone and ethyl pyruvate. This is subjected to coupling with 1‐O‐acetyl‐2,3,5‐tri‐O‐benzoyl‐β‐D‐ribofuranose under Vorbrüggen glycosylation conditions to provide the corresponding nucleoside in high yield. Activation of the nucleoside to the corresponding 2‐methylthio derivative followed by treatment with hydroxylamine hydrochloride in pyridine provides the corresponding 2‐hydroxyimino derivative in high yield. Finally, the synthesis of five free modified nucleoside analogs is described. The newly synthesized nucleosides have been evaluated against an RNA viral panel and moderate activity was observed against hepatitis C virus, Zika virus, and human respiratory syncytial virus. © 2021 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of 6‐aza‐5‐methyl‐2‐thiouracil

Basic Protocol 2: Preparation of 6‐aza‐5‐methyl‐2‐thiouridine and 6‐aza‐5‐methyluridine

Basic Protocol 3: Preparation of 6‐aza‐2‐hydroxyimino‐5‐methyluridine

Basic Protocol 4: Preparation of 6‐aza‐2‐hydroxyimino‐5‐methyl‐4‐thiouridine and 6‐aza‐2‐hydroxyimino‐5‐methylcytosine

A new family of hydrazone modified cytidine phosphoramidite building block was synthesized and incorporated into oligodeoxynucleotides to construct photoswitchable DNA strands. TheE‐Zisomerization triggered by the irradiation of blue light with a wavelength of 450 nm was investigated and confirmed by¹H NMR spectroscopy and HPLC in the contexts of both nucleoside and oligodeoxynucleotide. The light activatedZform isomer of this hydrazone‐cytidine with a six‐member intramolecular hydrogen bond was found to inhibit DNA synthesis in the primer extension model by usingBstDNA polymerase. In addition, the hydrazone modification caused the misincorporation of dATP together with dGTP into the growing DNA strand with similar selectivity, highlighting a potential G to A mutation. This work provides a novel functional DNA building block and an additional molecular tool that has potential chemical biology and biomedicinal applications to control DNA synthesis and DNA‐enzyme interactions using the cell friendly blue light irradiation.

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides

Basic Protocol 2: Digestion of RNA samples extracted from cells

Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry