Lihe Zhang

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

CD38/cADPR/Ca2+ pathway promotes cell proliferation and delays nerve growth factor-induced differentiation in PC12 cells.

Intracellular Ca2+ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca2+ changes. Here we explored the role of one endogenous Ca2+-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca2+ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca2+ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

A novel fluorescent cell membrane permeable caged cyclic ADP-Ribose analogue

Background: The available agonists for cADPR, an endogenous Ca2+-mobilizing nucleotide, are either weak or not cell-permeant. Results: We synthesized a coumarin-caged isopropylidene-protected cIDPRE (Co-i-cIDPRE), which is a potent and cell-permeant cADPR agonist. Conclusion: Uncaging of Co-i-cIDPRE activates RyRs for Ca2+ mobilization and triggers Ca2+ influx via TRPM2. Significance: Co-i-cIDPRE should provide a valuable tool to study cADPR/Ca2+ signaling. Cyclic adenosine diphosphate ribose is an endogenous Ca2+ mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca2+ increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca2+ release from endoplasmic reticulum, with concomitant Ca2+ influx in Jurkat cells. Ca2+ release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca2+ influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca2+ release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca2+ release from endoplasmic reticulum via RyRs and triggers Ca2+ influx via TRPM2.

A solid-phase method for peptide–siRNA covalent conjugates based on click chemistry

In order to prepare a new delivery system by chemical modification, peptide–siRNA conjugates have been obtained by a solid-phase click chemistry strategy, using an alkynyl nucleoside analogue “clicked” onto a peptide-derivatized CPG (Controlled Pore Glass) followed by oligonucleotides synthesis. The 3′-sense strand conjugate maintained good gene silencing activity, while that of the 3′-antisense strand conjugate decreased somewhat.

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.

Generation of shRNA Pool Library: A Revision of the Biological Technique from the Viewpoint of Chemistry

RNA interference (RNAi) is the process of using a small doublestranded RNA (siRNA) to knock down the expression level of sequence–homologue genes. The selective and robust effect of siRNA on gene expression makes RNAi one of the most important technological breakthroughs in modern scientific history. Short hairpin siRNA (shRNA) is the most widely used form of expressed siRNA in which RNA duplexes are tethered through a small loop. Currently the use of individual shRNA to study gene function is commonplace. The newest trend is to use siRNA/shRNA libraries to perform reverse-genetic screens of hundreds or even thousands of different genes in a single experiment. Three major factors limit the broad application of syntheticarray-based siRNA libraries: the enormous cost of genomewide siRNA libraries and the expensive robotic liquid handling systems for high-throughput screening as well as the difficulties in performing transfections reproducibly. A much easier alternative is to prepare an siRNA library that is first expressed in transduced cells, then the cells that have the desired phenotype are selected, and the siRNA sequence that is contained within the selected cells is identified. Several groups have reported a technique for the construction of such a shRNA pool library. The key step in this technique is to convert a pool of single-stranded hairpin DNA into a pool of doublestranded DNA (shRNA templates) via special DNA polymerase-catalyzed primer extension (Figure 1A) prior to cloning into expression vectors (see Figure S1 in the Supporting Information). Given the enormous advantage of this method, however, no follow-up application has been ACHTUNGTRENNUNGreported. Here, we revise the feasibility of this step from the viewpoint of chemistry. The goal of primer extension is to disrupt intramolecular base pairs within the highly stable hairpin DNA template, and to synthesize a complementary single-stranded DNA as shown in Figure 1A. It should be noted that the newly synthesized single-stranded DNA is not only complementary, but more importantly, sequence-identical to its template, and thus their annealing product is a palindromic DNA, except for the small loop portion. However, the fundamental concern is whether this reaction, that is, the displacement of intramolecular base pairs with intermolecular base pairs, is energetically favorable, or which form in this conversion, hairpin or the double strand, is more stable. In order to clarify this point, we tested the thermostability of palindromic DNA. A pool of double-stranded DNA (the siRNA-

Concise synthesis of novel acyclic analogues of cADPR with an ether chain as the northern moiety

To study the properties of hydrolysates of cyclic adenosine diphosphate ribose (cADPR), a series of novel acyclic analogues of cADPR with an ether chain as the northern moiety and 8-substituted adenine or hypoxanthine as the base moiety were synthesized via an N1 substitution construction, followed by bisphosphorylation, phosphoramidition or pyrophosphorylation. These compounds also provide various precursors for synthesizing cADPR analogues.