In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation.

Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.

Identification of a Tricyclic PIII Chiral Auxiliary for Solid-Supported Synthesis of Stereopure Phosphorothioate-Containing Oligonucleotides.

Since the recognition of oligonucleotides as a therapeutic modality, significant work has been devoted to improving therapeutic properties, including nuclease stability. Phosphorothioate (PS) modifications of phosphodiesters are one of the most explored chemical modification and integral to currently approved oligonucleotide therapeutics, including antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs). Insertion of sulfur into the phosphate bridge in an n-mer leads to 2n isomeric mixtures of PSs, with different nuclease stability and protein-binding properties. Efforts to create stereopure PS-containing oligonucleotides has spurred interest in identifying new synthetic methods. Herein, work on a novel and practical tricyclic PIII chiral auxiliary and its application in solid-supported synthesis of stereopure PS-containing oligonucleotides is reported.

Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition.

Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient methods for the attachment of a variety of reporter groups is Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust conjugation method is not yet established for these ONs due to a general concern of Cu-S interaction. Here, we developed a method allowing for efficient conjugation of peptides to PS oligonucleotides. The method utilizes solid supported oligonucleotides that can be readily transformed into "clickable ONs" by simple linker conjugation and further reacted with an azido containing moiety (e.g., a peptide) using the CuBr × Me2S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotide-conjugates by means of efficient Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition.

Homopurine RP-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2'-OMe)-RNAs.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester.

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0-8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39-85% yield. This method has potential uses in biotechnology and chemical biology.

Solid-Phase Synthesis of Phosphorothioate/Phosphonothioate and Phosphoramidate/Phosphonamidate Oligonucleotides.

We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.

Oligonucleotide⁻Palladacycle Conjugates as Splice-Correcting Agents.

2'-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5'-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents.

A Versatile and Convenient Synthesis of 34 S-Labeled Phosphorothioate Oligonucleotides.

A synthetic protocol for 34 S-labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS-based assay analysis. This was enabled by a highly efficient, two-step, one-pot synthesis of 34 S-labeled phenylacetyl disulfide (34 S-PADS), starting from 34 S-enriched elemental sulfur (34 S8 ). 34 S-PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The 34 S-SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled 32 S PS ONs.

Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping.

Oligonucleotides containing phosphorothioate (PS) linkages have recently demonstrated significant clinical utility. PS oligonucleotides are manufactured via a solid-phase chain elongation process in which a four-reaction cycle consisting of detritylation, coupling, sulfurization, and failure sequence capping with Ac2O is repeated. In the capping step, uncoupled sequences are acetylated at the 5'-OH to stop the chain growth and control the levels of deletion, or ( n-1), impurities. Herein, we report that the byproducts of commonly used sulfurization reagents react with the 5'-OH and cap the failure sequences. The standard Ac2O capping step can therefore be eliminated, and this 3-reaction cycle process affords a higher yield and higher or comparable overall purity compared to the conventional 4-reaction synthesis. This improvement results in reducing the number of reactions from ∼80 to ∼60 for the synthesis of a typical length 20-mer oligonucleotide. For every kilogram of an oligonucleotide intermediate synthesized, > 500 L of reagents and organic solvents is saved, and the E-factor is decreased to <1500 from ∼2000.

Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver.

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.

Phosphate-modified analogues of m(7)GTP and m(7)Gppppm(7)G-Synthesis and biochemical properties.

The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between ß and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.

Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine and 2'-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

DNA-mediated electron transfer in DNA duplexes tethered to gold electrodes via phosphorothioated dA tags.

The efficiency of DNA-based bioelectronic devices strongly depends on the way DNA molecules are linked to the electronic component. Commonly, DNA is tethered to metal electrodes via an alkanethiol linker representing an additional barrier for electron transport. Here we demonstrate that the replacement of the alkanethiol linker for a phosphorothioated adenosine tag increases the rate of DNA-mediated electron transfer (ET) up to 259 s(-1), representing the highest hitherto reported rate of electrochemically-modulated ET, and improves the stability of DNA-electrode surface binding. Both results offer pronounced technological and scientific benefits for DNA-based electronics.

Terminally modified, short phosphorothioate oligonucleotides as inhibitors of gene expression in cells.

Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5'- and 3'-modifications include 5'-trimethoxystilbene with 3'-uracil(anthraquinone)-cap, 5'-chloic acid derivative with 3'-uracyl(anthraquinone)-cap and 5'-cholic acid derivative with three 3'-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5'-cholic acid derivative with three 3'-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.

Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo.

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks. The degenerate NAP REP 2006 prevented the development of widespread and persistent DHBV infection in 14-day-old ducks, while the acidic-pH-sensitive NAP REP 2031 had little or no prophylactic effect. REP 2006 displayed significant toxicity in ducks, which was attributed to CpG-mediated proinflammation, while REP 2031 (which has no CpG motifs) displayed no toxicity. A third NAP, REP 2055, which was designed to retain amphipathic activity at acidic pH and contained no CpG motifs, was well tolerated and displayed prophylactic activity against DHBV infection at doses as low as 1 mg/kg of body weight/day. These studies suggest that NAPs can be easily and predictably tailored to retain anti-DHBV activity and to have minimal toxic effects in vivo. Future studies are planned to establish the therapeutic efficacy of NAPs against persistent DHBV infection.

Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.

Nucleic acid polymers (NAPs) utilize the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) to target protein interactions involved in viral replication. NAPs are broadly active against a diverse range of enveloped viruses that use type I entry mechanisms. The antiviral activity of NAPs against hepatitis B virus (HBV) infection was assessed in vitro in duck hepatitis B virus (DHBV)-infected primary duck hepatocytes (PDH). NAPs efficiently entered PDH in the absence of any transfection agent and displayed antiviral activity at concentrations of 0.01 to 10 µM, measured by their ability to prevent the intracellular accumulation of DHBV surface antigen, which was independent of their nucleotide sequence and was specifically dependent on phosphorothioation. Higher levels of antiviral activity were observed with NAPs 40 nucleotides in length or longer. The fully degenerate NAP (REP 2006) was active during DHBV infection or when added 12 h after infection. In contrast, an acidic-pH-sensitive NAP (REP 2031) that was broadly active against other viruses displayed antiviral activity when present during DHBV infection but no activity when added 12 h after infection, suggesting that NAPs exert their postentry effect in an acidic environment unique to DHBV infection. Both REP 2006 and REP 2031 displayed negligible cytotoxicity in PDH at concentrations of up to 10 µM, as assessed using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cytotoxicity assay. The antiviral activity of NAPs against DHBV in vitro was strictly dependent on their amphipathic character, suggesting that NAPs interact with amphipathic target(s) that are important for DHBV entry and postentry mechanisms required for infection.

Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts.

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.

Synthesis, properties, and applications of oligonucleotides containing an RNA dinucleotide phosphorothiolate linkage.

RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3'- or 5'-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage. Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3'- or 5'-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3'- or 5'-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3'- and 5'-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities.