Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer.

BACKGROUND: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. METHODS: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. RESULTS: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. CONCLUSIONS: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Structure of HIV-1 RT/dsRNA initiation complex prior to nucleotide incorporation.

The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) Nature 557:118-122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3' end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3' end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA-tRNALys3 in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.

Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV.

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5-10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT's activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

Non-nucleoside Reverse Transcriptase Inhibitors Inhibit Reverse Transcriptase through a Mutually Exclusive Interaction with Divalent Cation-dNTP Complexes.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are considered noncompetitive inhibitors that structurally alter reverse transcriptase (RT) and dramatically decrease catalysis. In this report, biochemical analysis with various divalent cations was used to demonstrate that NNRTIs and divalent cation-dNTP complexes are mutually exclusive, inhibiting each other's binding to RT/primer/template (RT-P/T) complexes. The binding of catalytically competent divalent cation-dNTP complexes to RT-P/T was measured with Mg2+, Mn2+, Zn2+, Co2+, and Ni2+ using Ca2+, a noncatalytic cation, for displacement. Binding strength order was Mn2+ ≈ Zn2+ ≫ Co2+ > Mg2+ ≈ Ni2+. Consistent with but not exclusive to mutually exclusive binding, primer extension assays showed that stronger divalent cation-dNTP complexes were more resistant to NNRTIs (efavirenz (EFV), rilpivirine (RPV), and nevirapine (NVP)). Filtration assays demonstrated that divalent cation-dNTP complexes inhibited the binding of 14C-labeled EFV to RT-P/T with stronger binding complexes formed with Mn2+ inhibiting more potently than those with Mg2+. Conversely, filter binding assays demonstrated that EFV inhibited 3H-labeled dNTP binding to RT-P/T complexes with displacement of Mn2+-dNTP complexes requiring much greater concentrations of EFV than the more weakly bound Mg2+-dNTP complexes. EFV bound relatively weakly to the NNRTI resistant K103N RT; but, binding was modestly enhanced in the presence of P/T, and EFV was easily displaced by divalent cation-dNTP complexes. This suggests that K103N overcomes EFV inhibition mostly by binding more weakly to the drug and is in contrast to other reports that indicate K103N has little to no effect on drug or dNTP binding. Overall, this biochemical analysis supports recent biophysical analyses of NNRTI-RT interactions that indicate mutually exclusive binding.

An Evolutionary/Biochemical Connection between Promoter- and Primer-Dependent Polymerases Revealed by Systematic Evolution of Ligands by Exponential Enrichment.

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified systematic evolution of ligands by exponential enrichment (SELEX) approach. Two Taq-specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. TaqI contained 8 nucleotides (5'-CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCRs. Similarly, exonuclease- Klenow polymerase also selected a high-affinity primer that contained a related core promoter sequence from phage T7 RNAP (5'-ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity, suggesting that binding was highly sequence specific. The results are discussed in the context of possible effects on multiprimer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs.IMPORTANCE This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function or be a consequence of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered, and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promoters. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology.

Physiological Mg2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro.

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.

Selection of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity.

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5' DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5' DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding.

Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro.

UNLABELLED: The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10(-4) range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity. However, cellular experiments suggest that HIV does not have significantly lower fidelity than other retroviruses and shows a mutation frequency in the 10(-5) range. In this report, we reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results from experiments conducted with physiological (for lymphocytes) concentrations of free Mg(2+) (~0.25 mM) and is comparable to Moloney murine leukemia virus (MuLV) RT fidelity. The physiological conditions produced mutation rates that were 5 to 10 times lower than those obtained under typically employed in vitro conditions optimized for RT activity (5 to 10 mM Mg(2+)). These results were consistent in both commonly used lacZα complementation and steady-state fidelity assays. Interestingly, although HIV RT showed severalfold-lower fidelity under high-Mg(2+) (6 mM) conditions, MuLV RT fidelity was insensitive to Mg(2+). Overall, the results indicate that the fidelity of HIV replication in cells is compatible with findings of experiments carried out in vitro with purified HIV RT, providing more physiological conditions are used. IMPORTANCE: Human immunodeficiency virus rapidly evolves through the generation and subsequent selection of mutants that can circumvent the immune response and escape drug therapy. This process is fueled, in part, by the presumably highly error-prone HIV polymerase reverse transcriptase (RT). Paradoxically, results of studies examining HIV replication in cells indicate an error frequency that is ~10 times lower than the rate for RT in the test tube, which invokes the possibility of factors that make RT more accurate in cells. This study brings the cellular and test tube results in closer agreement by showing that HIV RT is not more error prone than other RTs and, when assayed under physiological magnesium conditions, has a much lower error rate than in typical assays conducted using conditions optimized for enzyme activity.

Alternative divalent cations (Zn²âº, Co²âº, and Mn²âº) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.

BACKGROUND: Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg(2+) is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn(2+), Co(2+), and Zn(2+) can also support catalysis. Although Zn(2+) supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn(2+) is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn(2+). METHODS: We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn(2+), Co(2+), and Zn(2+). RESULTS: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3' nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn(2+) than Mg(2+). In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn(2+) and Co(2+) remained similar to Mg(2+) at lower concentrations that are optimal for catalysis. CONCLUSION: This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.

Synthetic, structural mimetics of the ß-hairpin flap of HIV-1 protease inhibit enzyme function.

Small-molecule mimetics of the ß-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 µM.

Inhibition of SARS-CoV-2 Infection in Human Airway Epithelium with a Xeno-Nucleic Acid Aptamer.

Background: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. Methods: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. Results: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. Conclusions: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.

Several physiologically relevant cations including Ca(2+), Mn(2+), and Zn(2+) have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg(2+) ions bound to RT. We analyzed the effects of Zn(2+) on reverse transcription and compared them to Ca(2+) and Mn(2+). Using nucleotide extension efficiency as a readout, Zn(2+) showed significant inhibition of reactions with 2 mM Mg(2+), even when present at only ∼5 µM. Mn(2+) and Ca(2+) were also inhibitory but at higher concentrations. Both Mn(2+) and Zn(2+) (but not Ca(2+)) supported RT incorporation in the absence of Mg(2+) with Mn(2+) being much more efficient. The maximum extension rates with Zn(2+), Mn(2+), and Mg(2+) were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 µM, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn(2+) and Mg(2+), and single RT molecules were able to continue extension in the presence of Zn(2+) for several hours on the same template. Consistent with this result, the half-life for RT-Zn(2+)-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg(2+), indicating ∼130-fold more stable binding with Zn(2+). Essentially, the presence of Zn(2+) promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.

Duplex structural differences and not 2'-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA.

Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3' recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2'- hydroxyls on pivotal RNA bases with 2'-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. RT's K(d) (k(off)/k(on)) for DNA-DNA and RNA-DNA were similar, although k(off) differed almost 40-fold, suggesting a faster k(on) for DNA-DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA-DNA may aid in degradation of RNA fragments that remain after DNA synthesis.

Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (KD,app) of 93 ± 7 nM. The dissociation rate constant (koff) and binding half-life (t1/2) for E9-R5-12 were 0.083 ± 0.019 min-1 and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3'-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and Taq and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.

Structural basis of HIV inhibition by L-nucleosides: Opportunities for drug development and repurposing.

Infection with HIV can cripple the immune system and lead to AIDS. Hepatitis B virus (HBV) is a hepadnavirus that causes human liver diseases. Both pathogens are major public health problems affecting millions of people worldwide. The polymerases from both viruses are the most common drug target for viral inhibition, sharing common architecture at their active sites. The L-nucleoside drugs emtricitabine and lamivudine are widely used HIV reverse transcriptase (RT) and HBV polymerase (Pol) inhibitors. Nevertheless, structural details of their binding to RT(Pol)/nucleic acid remained unknown until recently. Here, we discuss the implications of these structures, alongside related complexes with L-dNTPs, for the development of novel L-nucleos(t)ide drugs, and prospects for repurposing them.

Improving aptamer performance with nucleic acid mimics: de novo and post-SELEX approaches.

Aptamers are structural single-stranded oligonucleotides generated in vitro to bind to a specific target molecule. Aptamers' versatility can be enhanced with nucleic acid mimics (NAMs) during or after a selection process, also known as systematic evolution of ligands by exponential enrichment (SELEX). We address advantages and limitations of the technologies used to generate NAM aptamers, especially the applicability of existing engineered polymerases to replicate NAMs and methodologies to improve aptamers after SELEX. We also discuss the limitations of existing methods for sequencing NAM sequences and bioinformatic tools to predict NAM aptamer structures. As a conclusion, we suggest that NAM aptamers might successfully compete with molecular tools based on proteins such as antibodies for future application.

Cryo-EM structure of the HIV-1 Pol polyprotein provides insights into virion maturation.

Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo-electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.

A primer-free method that selects high-affinity single-stranded DNA aptamers using thermostable RNA ligase.

This article describes a method for selecting single-stranded DNA (ssDNA) molecules that bind with high-affinity aptamers to specific target proteins. This SELEX (systematic evolution of ligands by exponential enrichment) method is similar to other "primer-free" approaches where the random sequence ssDNA starting pool has no fixed sequences at the 5' and 3' termini. Therefore, there are no predetermined sequences that could bias selection. Like other SELEX methods, repeated cycles (typically 5-15) of selection and then amplification and reselection are used. The method differs from other primer-free approaches in that the key step for regenerating new material for subsequent rounds is ligation of the selected ssDNA to a defined sequence oligonucleotide using thermostable RNA ligase. Under specific conditions, this ligase ligated 30-nt random sequence ssDNA (5'-N(30)-3') to a specified 20-nt ssDNA with approximately 50% efficiency. Efficiency was improved to approximately 90% by the addition of a single T residue to the 3' end (5'-N(29)T-3'). High efficiency in this step is critical, especially early in the procedure because any selected material that is not ligated is lost. In this study, human immunodeficiency virus reverse transcriptase was used as the target protein, but the method could be applied to essentially any protein.

Xeno-Nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding.

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

HIV Reverse Transcriptase Pre-Steady-State Kinetic Analysis of Chain Terminators and Translocation Inhibitors Reveals Interactions between Magnesium and Nucleotide 3'-OH.

Deoxythymidine triphosphate analogues with various 3' substituents in the sugar ring (-OH (dTTP)), -H, -N3, -NH2, -F, -O-CH3, no group (2',3'-didehydro-2',3'-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3'-OH but with 4' additions (4'-C-methyl, 4'-C-ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (k obs) compared to dTTP ranging from about 2-fold (3'-H, -N3, and d4TTP with high Mg2+) to >10-fold (3'-NH2 and 3'-F with low Mg2+), while 3'-O-CH3 dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg2+ and the 3'-OH, k obs using 5 µM dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogues with 3' alterations incorporated 2.8-5.1-fold slower in 0.5 mM Mg2+. In contrast, 4'-C-methyl and d-carba dTTP, which retain the 3'-OH, were not significantly affected by Mg2+. Consistent with these results, analogues with 3' modifications were better inhibitors in 6 versus 0.5 mM Mg2+. Equilibrium dissociation constant (K D) and maximum incorporation rate (k pol) determinations for dTTP and analogues lacking a 3'-OH indicated that low Mg2+ caused a several-fold greater reduction in k pol with the analogues but did not significantly affect K D, results consistent with a role for 3'-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg2+ and the 3'-OH of the incoming nucleotide and suggest that inhibitors with 3'-OH groups may have advantages in low free Mg2+ in physiological settings.