Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A.

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH2 group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

Understanding intercalative modulation of G-rich sequence folding: solution structure of a TINA-conjugated antiparallel DNA triplex.

We present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.

The APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis promotes viral and cancer evolution, thus enabling the progression of diseases and development of drug resistance. Therefore, APOBEC3 inhibition offers a possibility to complement existing antiviral and anticancer therapies and prevent the emergence of drug resistance, thus making such therapies effective for longer periods of time. Here, we synthesised nucleosides containing seven-membered nucleobases based on azepinone and compared their inhibitory potential against human cytidine deaminase (hCDA) and APOBEC3A with previously described 2'-deoxyzebularine (dZ) and 5-fluoro-2'-deoxyzebularine (FdZ). The nanomolar inhibitor of wild-type APOBEC3A was obtained by the incorporation of 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one in the TTC loop of a DNA hairpin instead of the target 2'-deoxycytidine providing a Ki of 290 ± 40 nM, which is only slightly weaker than the Ki of the FdZ-containing inhibitor (117 ± 15 nM). A less potent but notably different inhibition of human cytidine deaminase (CDA) and engineered C-terminal domain of APOBEC3B was observed for 2'-deoxyribosides of the S and R isomers of hexahydro-5-hydroxy-azepin-2-one: the S-isomer was more active than the R-isomer. The S-isomer shows resemblance in the position of the OH-group observed recently for the hydrated dZ and FdZ in the crystal structures with APOBEC3G and APOBEC3A, respectively. This shows that 7-membered ring analogues of pyrimidine nucleosides can serve as a platform for further development of modified ssDNAs as powerful A3 inhibitors.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A1-4 is an endogenous mutagen in many different human cancers5-7, where it becomes hijacked to fuel tumor evolvability. APOBEC3A's single-stranded DNA C-to-U editing activity1,8 results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations5-7. Transgenic expression in mice demonstrates its tumorigenic potential9. APOBEC3A inhibitors may therefore comprise a novel class of anti-cancer agents that work by blocking mutagenesis, preventing tumor evolvability, and lessening detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC recognition motif that is part of a three-nucleotide loop. The nuclease-resistant phosphorothioated derivatives of these inhibitors maintain nanomolar in vitro potency against APOBEC3A, localize to the cell nucleus, and block APOBEC3A activity in human cells. These results combine to suggest roles for these inhibitors to study A3A activity in living cells, potentially as conjuvants, leading toward next-generation, combinatorial anti-mutator and anti-cancer therapies.

Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.

Phosphorothioate modification improves exon-skipping of antisense oligonucleotides based on sulfonyl phosphoramidates in mdx mouse myotubes.

2'-O-Methyl (2'-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2'-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2'-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications.

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies.

Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside ß-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

The Importance of Phosphates for DNA G-Quadruplex Formation: Evaluation of Zwitterionic G-Rich Oligodeoxynucleotides.

A quaternary ammonium butylsulfonyl phosphoramidate group (N+) was designed to replace all the phosphates in a G-rich oligodeoxynucleotide d(TG4 T), resulting in a formally charge-neutral zwitterionic N+TG4 T sequence. We evaluated the effects of N+phosphate modifications on the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes (G4) formed by TG4 T and compared them to the properties of the recently published phosphoryl guanidine d(TG4 T) (PG-TG4 T). Using size-exclusion chromatography, we established that, unlike PG-TG4 T, which exists as a mixture of complexes of different molecularity in solution, N+TG4 T forms an individual tetramolecular complex. In contrast to PG modifications that destabilized G4s, the presence of N+ modifications increased thermal stability relative to unmodified [d(TG4 T)]4 . The initial stage of assembly of N+TG4 T proceeded faster in the presence of Na+ than K+ ions and, similarly to PG-TG4 T, was independent of the salt concentration. However, after complex formation exceeded 75 %, N+TG4 T in solution with Na+ showed slower association than with K+ . N+TG4 T could also form G4s in solution with Li+ ions at a very low strand concentration (10 µM); something that has never been reported for the native d(TG4 T). Charge-neutral PG-G4s can invade preformed native G4s, whereas no invasion was observed between N+and native G4s, possibly due to the increased thermal stability of [N+TG4 T]4 . The N+ modification makes d(TG4 T) fully resistant to enzymatic digestion, which could be useful for intracellular application of N+-modified DNA or RNA.

Heterochromatin protein 1α interacts with parallel RNA and DNA G-quadruplexes.

The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4-DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome.

Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3GCTD and in that of full-length wild-type APOBEC3G.

Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2'-deoxyzebularine.

To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As such, APOBEC3B promotes evolution and progression of cancers and leads to development of drug resistance in multiple cancers. As APOBEC3B is a non-essential protein, its inhibition can be used to suppress emergence of drug resistance in existing anti-cancer therapies. Because of the vital role of APOBEC3 enzymes in innate immunity, selective inhibitors targeting only APOBEC3B are required. Here, we use the discriminative properties of wild-type APOBEC3A, APOBEC3B and APOBEC3G to deaminate different cytosines in the CCC-recognition motif in order to best place the cytidine analogue 2'-deoxyzebularine (dZ) in the CCC-motif. Using several APOBEC3 variants that mimic deamination patterns of wild-type enzymes, we demonstrate that selective inhibition of APOBEC3B in preference to other APOBEC3 constructs is feasible for the dZCC motif. This work is an important step towards development of in vivo tools to inhibit APOBEC3 enzymes in living cells by using short, chemically modified oligonucleotides.

Effects of Pressure and pH on the Physical Stability of an I-Motif DNA Structure.

The thermodynamic stability of a cytosine(C)-rich i-motif tract of DNA, which features pH-sensitive [C..H..C]+ moieties, has been studied as function of both pressure (0.1-200 MPa) and pH (3.7-6.2). Careful attention was paid to correcting citrate buffer pH for known variations that stem from changes in pressure. Once pH-corrected, (i) at pH >4.6 the i-motif becomes less stable as pressure is increased (KD decreases), giving a small negative volume change for dissociation (ΔD V°) of the i-motif - a conclusion opposite to that which would be drawn if the buffer pH was not corrected for the effects of pressure; (ii) the i-motif's melting temperature increases by more than 30 K between pH 6.5 and 4.5, the consequence of an enthalpy for dissociation (ΔD H°) of 77(3) and 90(3) kJ (mol H+ )-1 at 0.1 and 200 MPa, respectively; (iii) below pH 4.6 at 0.1 MPa (pH 4.3 at 200 MPa) the melting temperature decreases as a result of double protonation of cytosine pairs, and ΔD H° and ΔD V° change signs; and (iv) the combination of ΔD H° and ΔD V° lead to the melting temperature at pH 4.3 being 3 K higher at 200 MPa than at 0.1 MPa.

α-2'-Deoxyguanosine can switch DNA G-quadruplex topologies from antiparallel to parallel.

Here we demonstrate G-quadruplex formation by oligodeoxynucleotides containing α-2'-deoxyguanosine (α-dG) as a sole source of guanosines in G4T4, G4T4G4 and T(G3Tn)3G3T sequences with various numbers of natural ß-T in the loops (n = 1-4). Based on circular dichroism spectra we observed that all α-dG-containing DNAs formed G-quadruplexes with uniform arrangement of α-dG-tetrads, which implies formation of G-quadruplexes of parallel topology. In several cases, native DNA structures that usually adopt an antiparallel topology were converted to more thermally stable G-quadruplexes of parallel topology. Using 2D ROESY NMR spectra a new 'sequential walk' was established for α-dGs in a tetramolecular, parallel complex formed by the α-G4ß-T4 sequence. Analysis of ROEs in α-dGs indicates that guanines in [α-G4ß-T4]4 adopt anti-glycosidic conformations. These results demonstrate that α-dG can be used for an antiparallel-to-parallel switch of G-quadruplex DNAs producing complexes with higher thermal stability and uniform stacking of α-dG-tetrads.

Neutral and Negatively Charged Phosphate Modifications Altering Thermal Stability, Kinetics of Formation and Monovalent Ion Dependence of DNA G-Quadruplexes.

The effect of phosphate group modifications on formation and properties of G-quadruplexes (G4s) has not been investigated in detail. Here, we evaluated the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes formed by oligodeoxynucleotides d(G4 T), d(TG4 T) and d(TG5 T), in which all phosphates were replaced with N-methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups resulting in either negatively charged or neutral DNA sequences, respectively. We established that all modified sequences were able to form G-quadruplexes of parallel topology; however, the presence of modifications led to a decrease in thermal stability relative to unmodified G4s. In contrast to negatively charged G4s, assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions, and was independent of the salt concentration used. Formation of mixed G4s composed of both native and neutral G-rich strands has been detected using native gel electrophoresis, size-exclusion chromatography and ESI-MS. In summary, our results indicate that the phosphate modifications studied are compatible with G-quadruplex formation, which could be used for the design of biologically active compounds.

Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2'-Deoxyzebularine Analogues.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2'-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.

NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate.

APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, but they are also involved in cancer evolution and development of drug resistance. We report a solution-state model of APOBEC3A interaction with its single-stranded DNA substrate obtained with the 'method of small changes'. This method compares pairwise the 2D 15N-1H NMR spectra of APOBEC3A bearing a deactivating mutation E72A in the presence of 36 slightly different DNA substrates. From changes in chemical shifts of peptide N-H moieties, the positions of each nucleotide relative to the protein can be identified. This provided distance restraints for molecular-dynamic simulations to derive a 3-D molecular model of the APOBEC3A-ssDNA complex. The model reveals that loops 1 and 7 of APOBEC3A move to accommodate substrate binding, indicating an important role for protein-DNA dynamics. Overall, our method may prove useful to study other DNA-protein complexes where crystallographic techniques or full NMR structure calculations are hindered by weak binding or other problems. Subsequent to submission, an APOBEC3A structure with a bound DNA oligomer was published and coordinates released, which has provided an unbiased validation of the 'method of small changes'.