Structure-guided development of Pb2+-binding DNA aptamers.

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.

Base Pairing and Functional Insights into N3-Methylcytidine (m3C) in RNA.

N3-methylcytidine (m3C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m3C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m3C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m3C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m3C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m3C using reverse transcription in vitro shows that N3-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m3C in RNA and a foundation for developing new molecular tools for mapping m3C in different RNA contexts and exploring the biochemical and biomedical potentials of m3C in the design and development of RNA based therapeutics.

Short DNA Oligonucleotide as a Ag+ Binding Detector.

Ag+ has been known to mediate several natural metallo-base pairs. Based on the unique structural information of a short 8-mer DNA strand (5'-GCACGCGC-3') induced by Ag+, we constructed several fluorescent DNA beacons for the detection of Ag+ according to the increase in the fluorescence emission on Ag+ binding. This Ag+ detection assay is quick, sensitive, and easy to adapt and can function in a wide range of temperatures from 5 to 65 °C.

Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA.

The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

High-resolution DNA quadruplex structure containing all the A-, G-, C-, T-tetrads.

DNA can form diverse structures, which predefine their physiological functions. Besides duplexes that carry the genetic information, quadruplexes are the most well-studied DNA structures. In addition to their important roles in recombination, replication, transcription and translation, DNA quadruplexes have also been applied as diagnostic aptamers and antidisease therapeutics. Herein we further expand the sequence and structure complexity of DNA quadruplex by presenting a high-resolution crystal structure of DNA1 (5'-AGAGAGATGGGTGCGTT-3'). This is the first quadruplex structure that contains all the internal A-, G-, C-, T-tetrads, A:T:A:T tetrads and bulged nucleotides in one single structure; as revealed by site-specific mutagenesis and biophysical studies, the central ATGGG motif plays important role in the quadruplex formation. Interestingly, our structure also provides great new insights into cation recognition, including the first-time reported Pb2+, by tetrad structures.

Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex.

5-Cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base-pairing stability and specificity studies in RNA duplexes indicated that cnm5 U slightly decreased the stability of the duplex but retained the base-pairing preference. In contrast, cn5 U dramatically decreased both base-pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn5 U:G pair was found to be stronger than the cn5 U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn5 U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn5 U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5' side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson-Crick A:U pair to a mismatched C:U pair in the 5'-neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron-withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA-based therapeutics.

A DNA Structure Containing AgI -Mediated G:G and C:C Base Pairs.

Metal-mediated base pairs have been extensively utilized in many research fields, including genetic-code extension, novel therapeutics development, and nanodevice design. Compared to other cations, AgI is more flexible in pairing with natural base pairs. Herein, we present a DNA structure containing two C-AgI -C pairs and the first reported G-AgI -G pair in a short 8mer DNA strand. This structure not only provides detailed insight into these AgI -mediated base-pairing patterns in DNA, but also represents the first nonhelical DNA structure driven by heavy-metal ions, thus further contributing to the structural diversity of DNA. This unique complex structure is highly sequence-dependent, thus implying functional potentials as a new DNA aptamer that can bind and recognize silver ions. These results not only advance our understanding of the interactions between AgI and nucleobases, but also provide a unique structural component for the rational design of new DNA nanodevices.

Structural insights into RNA duplexes with multiple 2΄-5΄-linkages.

2΄-5΄-linked RNAs play important roles in many biological systems. In addition, the mixture of 2΄-5΄ and 3΄-5΄ phosphodiester bonds have emerged as a plausible structural element in prebiotic RNAs. Toward our mechanistic studies of RNA folding and structures with heterogeneous backbones, we recently reported two crystal structures of a decamer RNA duplex containing two and six 2΄-5΄-linkages, showing how RNA duplexes adjust the structures to accommodate these non-canonical linkages (Proc. Natl. Acad. Sci. USA, 2014, 111, 3050-3055). Herein, we present two additional high-resolution crystal structures of the same RNA duplex containing four and eight 2΄-5΄-linkages at different positions, providing new insights into the effects of these modifications and a dynamic view of RNA structure changes with increased numbers of 2΄-5΄-linkages in the same duplex. Our results show that the local structural perturbations caused by 2΄-5΄ linkages can be distributed to nearly all the nucleotides with big ranges of changes in different geometry parameters. In addition, hydration pattern and solvation energy analysis indicate less favorable solvent interactions of 2΄-5΄-linkages comparing to the native 3΄-5΄-linkages. This study not only promotes our understanding of RNA backbone flexibility, but also provides a knowledge base for studying the biochemical and prebiotic significance of RNA 2΄-5΄-linkages.

Multiplexed Activity of perAuxidase: DNA-Capped AuNPs Act as Adjustable Peroxidase.

In this study, we have investigated the intrinsic peroxidase-like activity of citrate-capped AuNPs (perAuxidase) and demonstrated that the nanozyme function can be multiplexed and tuned by integrating oligonucleotides on a nanoparticle surface. Systematic studies revealed that by controlling the reaction parameters, the mutiplexing effect can be delayed or advanced and further used for aptasensor applications.

Water-bridged hydrogen bond formation between 5-hydroxylmethylcytosine (5-hmC) and its 3'-neighbouring bases in A- and B-form DNA duplexes.

5-Hydroxylmethylcytosine (5hmC) has been recognized as the sixth base with important biological functions in many tissues and cell types. We present here the high-resolution crystal structures and molecular simulation studies of both A-form and B-form DNA duplexes containing 5hmC. We observed that 5hmC interacts with its 3'-neighboring bases through water-bridged hydrogen bonds and these interactions may affect the further oxidation of 5hmC.

Synthesis and base pairing studies of geranylated 2-thiothymidine, a natural variant of thymidine.

The synthesis and base pairing of DNA duplexes containing the geranylated 2-thiothymidine have been investigated. This naturally existing hydrophobic modification could grant better base pairing stability to the T-G pair over normal T-A and other mismatched pairs in the duplex context. This study provides a potential explanation for the different codon recognition preferences of the geranylated tRNAs.

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia.

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.