Borrelia burgdorferi exhibits dramatic changes in gene expression as it transits between its tick vector and vertebrate host. A major hurdle to understanding the mechanisms underlying gene regulation in B. burgdorferi has been the lack of a functional assay to test how gene regulatory proteins and sigma factors interact with RNA polymerase to direct transcription. To gain mechanistic insight into transcriptional control in B. burgdorferi, and address sigma factor function and specificity, we developed an in vitro transcription assay using the B. burgdorferi RNA polymerase holoenzyme. We established reaction conditions for maximal RNA polymerase activity by optimizing pH, temperature, and the requirement for divalent metals. Using this assay system, we analyzed the promoter specificity of the housekeeping sigma factor RpoD to promoters encoding previously identified RpoD consensus sequences in B. burgdorferi. Collectively, this study established an in vitro transcription assay that revealed RpoD-dependent promoter selectivity by RNA polymerase and the requirement of specific metal cofactors for maximal RNA polymerase activity. The establishment of this functional assay will facilitate molecular and biochemical studies on how gene regulatory proteins and sigma factors exert control of gene expression in B. burgdorferi required for the completion of its enzootic cycle.The Lyme disease spirochete  Borrelia burgdorferi is a highly fastidious host-associated bacterium in the phylum Spirochaetes2. B. burgdorferi is adapted to a vector-host life cycle and possesses a condensed 1.3 Mb genome with limited metabolic capability4. The genome lacks genes encoding components of the citric acid cycle, the electron transport chain, amino acid biosynthesis, and fatty acid biosynthesis pathways, wholly relying on the transport of sugars, fatty acids and amino acids from the environment for survival6. B. burgdorferi growth occurs extracellularly in vertebrate tissues and in ticks following a blood meal, which poses additional nutritional constraints7. In particular, the host competition for iron is hypothesized to have resulted in abstinence from iron utilization by B. burgdorferi10. Instead, manganese is thought to largely replace iron as a metal cofactor for metabolic enzymes11.B. burgdorferi regulates gene expression to adapt to environmental constraints faced in its enzootic cycle. B. burgdorferi responds to environmental changes in pH, temperature, nutrient availability, and manganese levels with dramatic shifts in transcription and growth18. The transcriptional changes in B. burgdorferi during transmission from the arthropod vector Ixodes scapularis20 are thought to be influenced by the mechanisms underlying environmental sensing and transcriptional responses. Mechanistic studies of how transcription factors regulate gene expression in B. burgdorferi have been hindered by the scarcity of biochemical tools. To understand the transcriptional mechanisms that support B. burgdorferi survival, we set out to characterize the RNA polymerase of B. burgdorferi.B. burgdorferi specific in vitro transcription assay, we purified intact RNA polymerase core enzyme, and the housekeeping sigma factor RpoD (σ70). Bacterial RNA polymerases are composed of four polypeptide subunits designated α, β, β′, and σ. The largest subunit β′ houses the active site that catalyzes RNA synthesis. In the cell, the β′ subunit is associated with the β and α subunits and forms the core enzyme composed of the two α subunits, one β subunit, and one β′ subunit that is capable of transcriptional elongation on single-stranded DNA21. The σ subunit is required for the recognition of the promoter and opening of the DNA double helix22. An additional, small subunit, ω, is also known to promote polymerase activity23. In this study, we characterized the subunit composition, reaction conditions, and sigma factor-dependent transcriptional activity of RNA polymerase purified from B. burgdorferi. We assayed previously annotated B. burgdorferi transcriptional start sites using RpoD to demonstrate the utility of this new in vitro transcription system.To develop a B. burgdorferi RNA polymerase core enzyme was generated to evaluate its structure for affinity chromatography purification. Individual models of β′, β, and α, subunits were generated with I-TASSER24 and aligned to a model of E. coli RNA polymerase (3lu0)25. An additional subunit, ω, which complexes with the RNA polymerase core at the carboxyl terminus of the β′ subunit, was included in the model to assess its influence on structure. Only minimal relaxation of the aligned model was necessary to prevent clash at the inter-protomer interfaces. Purification of an active RNA polymerase core from other bacteria has been achieved through affinity chromatography strategies that result in co-purification of the RNA polymerase protein complex26. Typically, the β′ subunit is appended with a carboxyl-terminal affinity-tag. The potential location of the β′ subunit carboxy-terminus is illustrated in Fig. B. burgdorferi strain harboring a sequence encoding a polyhistidine-tag on the 3′ end of rpoC (encoding the RNA polymerase β′ subunit) by homologous recombination and designated the strain 5A4-RpoC-His10X.A model of the B. burgdorferi RNA polymerase would co-purify with the affinity-tagged β′ subunit, 3–4 L cultures of 5A4-RpoC-His10X were grown to mid-logarithmic growth phase (~5 × 107 spirochetes · ml−1), and cell lysates generated using a French pressure cell were subjected to nickel-metal affinity chromatography. The presence of RNA polymerase in the elution fractions was analyzed by SDS-PAGE and staining of proteins with Coomassie Brilliant Blue. Elution fractions containing RNA polymerase were pooled and yielded 40–50 µg of extracted protein. The components of the RNA polymerase holoenzyme  in the pooled elution fractions were identified by LC-MS following separation of proteins by SDS-PAGE and in-gel digestion , zinc chloride, calcium chloride, and/or manganese chloride (MnCl2) to a base buffer containing a final concentration of 40 mM HEPES, pH 7.5, 0.05% NP40, and 1 mM DTT. Initial in vitro transcription reaction mixtures containing 100 nM DNA template, 100 nM RNA polymerase, and 200 µM nucleotides (NTPs) were incubated for 5–6 hours at 37 °C to allow for the accumulation of RNA transcripts . Our data indicated reactions containing 12 mM MgCl2 or 2–10 mM MnCl2 had detectable levels of RNA product accumulation based on SYBR Safe dye incorporation suggesting a metal requirement for RNA polymerase enzymatic activity. Further characterization determined RNA products were detectable within minutes when RNA polymerase was pre-incubated with 2–10 mM MnCl2 prior to addition of the DNA template to initiate the reaction. Collectively, these experiments established initial reaction conditions for achieving enzymatic activity from affinity-purified RNA polymerase. Subsequent reactions to test RNA polymerase activity from dsDNA templates were carried out in 60 mM potassium glutamate, 2 mM MgCl2, and 5 mM MnCl2 in addition to the base buffer described above.To determine reaction conditions that support RNA polymerase activity, we utilized a dye-incorporation method for detection of RNA synthesis that allowed for various reaction conditions to be screenedflgB promoter (flgBp) from B. burgdorferi genomic DNA by PCR. The flgB promoter is RpoD-dependent32 and the 499-bp PCR product encompasses a region from −248 to +251 surrounding the transcription initiation site. Levels of RpoD required for RNA polymerase activity initiated from the flgB promoter were determined by titration of recombinant RpoD in the reactions  were tested at 30 °C , 0–20 mM manganese sulfate (MnSO4), or 2 mM MgSO4 with 0–20 mM MnSO4 were prepared in parallel  manganese for detectable activity and did not require magnesium ions in the reaction mixture. Reaction mixtures containing 2 mM MgSO4 along with varying levels of MnSO4 all had higher levels of activity compared to reaction mixtures with only MnSO4 . Therefore, we screened the metal-dependent activity of RNA polymerase holoenzyme with recombinant RpoD, using the dsDNA ate Fig. . We testlel Fig. . The accSO4 Fig. . These rB. burgdorferi RNA polymerase β′ subunit was aligned to β′ subunits from other bacterial species to better understand the role of manganese using Clustal Omega and MSAProbs amino acid sequence alignment algorithms utilizing hidden Markov models38. Gram positive species in the genera Bacillus and Clostridium possess RNA polymerases that are enhanced by the presence of manganese40. The β′ subunits of these bacteria were included in the alignment to the β′ subunit in B. burgdorferi, along with prototypical magnesium-dependent RNA polymerases from other genera. However, the alignment around the active site, which binds magnesium, revealed no conserved pattern among the manganese-associating RNA polymerases  using previously annotated transcription initiation sites  in our in vitro transcription system.We next tested if the RNA polymerase holoenzyme can select various promoter sites encoded within the ity Fig. . Reactiotes Fig. . ConservB. burgdorferi by affinity chromatography, and an in vitro transcription assay was developed by establishing buffer and metal concentrations for reaction mixtures. The activity of RNA polymerase in a range of pH and temperature was demonstrated. We leveraged this in vitro transcription system to assay RpoD promoter selection on previously annotated promoters. These promoter sites were recognized by RpoD, as evidenced by specific length products produced by the RNA polymerase. This demonstrates the utility of these transcriptional start sites previously assayed and annotated in a 5′ end transcriptome and makes possible an assay of RpoD-dependent transcriptional initiation.In this study, an intact RNA polymerase complex was purified from in vitro transcription assays does not necessarily closely correlate with relative strength of transcriptional start sites measured from live cells. For example, ospAp, clpCp, and glpFp all produce in vitro transcription products of similar signal intensity . While prototypical bacterial RNA polymerases are thought to be magnesium-dependent51, RNA polymerases purified from the Firmicutes  are manganese-dependent53. A regulatory role for manganese has been demonstrated for some previously characterized RNA polymerases, such as transcription stalling, while others seem to require manganese for catalytic activity56. Although the highest level of activity from the B. burgdorferi RNA polymerase was obtained from a combination of magnesium and manganese, the role of manganese in catalysis remains unexplored; note that the conserved catalytic site amino acid residues do not differ between magnesium- and manganese-utilizing bacteria. Moreover, the possibility that metals such as magnesium may co-purify with the RNA polymerase was not ruled out. Further structural characterization is required to determine the exact roles of manganese and magnesium for B. burgdorferi RNA polymerase activity.In this study, we determined that RNA polymerase of B. burgdorferi physiology is its accumulation of manganese in the intracellular environment along with the apparent lack of intracellular iron10. The role of manganese in the B. burgdorferi RNA polymerase should be carefully considered due to its numerous potential roles in physiology. Manganese levels in B. burgdorferi are maintained by the BmtA transporter58, and spirochetes actively accumulate manganese in the cytosol. Levels of intracellular manganese in B. burgdorferi can change depending on the manganese concentration in the culture media57. Additionally, manganese levels were shown to affect the transcription of virulence genes and are hypothesized to be an environmental cue required for B. burgdorferi transmission59. The impact of varying concentrations of manganese on RNA polymerase activity in this study suggests regulation of optimal intracellular levels of manganese in B. burgdorferi are required to support basal transcription.A unique feature of B. burgdorferi. These transcription factors include Rrp2, BosR (the ferric uptake regulator homolog), BadR (the Borrelia host adaptation regulator), and DksA66. Our newly developed in vitro transcription assay can be used to confirm the function of these factors as activators or repressors of transcription from cognate promoters, which was not previously feasible for B. burgdorferi. Additionally, we observed RpoD only weakly drove transcriptional initiation from the ospC promoter, which is known to be regulated by the alternative sigma factor RpoS68. The data presented here demonstrate the utility of in vitro transcription assays to test the hypothesis that alternative sigma factors control transcription of dissimilar sets of genes in B. burgdorferi.Currently, several transcription factors are proposed to respond to environmental cues and to regulate transcription initiation in 69. An alignment of the resulting I-TASSER models to the corresponding subunits of a molecular model of the E. coli RNA polymerase core deposited in the Protein Databank under accession number 3lu025 was then performed with PyMOL . To relax the structure and relieve any clashes arising from the piece-wise modelling and assembly approach taken to generating the B. burgdorferi RNA polymerase core model, the assembled complex was subjected to minimization and limited molecular dynamics simulation with the program NAMD70 using the CHARMM36m force field71. The model was solvated with TIP3P waters and NaCl added to 250 mM using VMD72. The system was then minimized using 5,000 steps of conjugate gradient minimization with NAMD ahead of 100 ps of dynamics with a 2 fs integration time step during which the protein was fixed. Langevin dynamics temperature and Nosé-Hoover Langevin piston pressure controls were used to maintain the system at 310 K and 1 atm, respectively, and electrostatic interactions were calculated using the particle-mesh Ewald method. The system was then subjected to a second round of 5,000 steps of conjugate gradient minimization prior to performing 1 ns of unconstrained dynamics in the NPT ensemble. Protein coordinates from frames written every 2 ps over the final 500 ps were positionally-averaged, and bond lengths and angles were subsequently idealized with Rosetta73 to generate the final model.Amino acid sequences of the RNA polymerase subunits  were individually submitted to the Iterative Threading Assembly Refinement (I-TASSER) server for modeling with no template specificationrpoC in the B31–5A4 strain background by homologous recombination. The 3′ end of the rpoC gene was amplified using primers encoding a 10xHis affinity tag optimized for B. burgdorferi codon usage (rpoC 3285 F: TGCATCTTATGTATTACCAG and rpoC 4131 R + H + AaAg: ACCGGTACTGACGTCTCACTAGTGATGATGATGGTGATGATGGTGATG ATGAACTTCAGAATCGATATTT). The PCR product was TOPO-cloned into the pCR2.1-TOPO vector . The genomic sequence downstream of rpoC was amplified by PCR using the primers rpsL U149F + AatII: GACGTCTGGACATTTAATTCCTACTG and rpsG 385 R + AgeI: ACCGGTATGCATTTAAAAGTTCGTTT. The PCR product encoding the downstream region and the pCR2.1-TOPO-RpoC-10XHis vector were digested by AatII and AgeI restriction enzymes and ligated together with T4 DNA ligase . The selectable marker flgBp-aaaC1-trpLt12 was inserted on the 3′ end of rpoC−10XHis. AatII restriction digest of the pCR2.1-TOPO-RpoC-10xHis vector, and the PCR product encoding the selectable marker was ligated with T4 ligase to generate the plasmid used for homologous recombination. B. burgdorferi B31–5A4 strain was transformed with pCR2.1-TOPO-RpoC-10xHis plasmid as described using 30 µg · ml−1 gentamicin for selection74.A C-terminal 10xHis affinity tag was introduced to the chromosomal copy of B. burgdorferi B31–5A4-RpoC-10xHis strain was maintained under microaerobic conditions  in BSK II medium, pH 7.6 at 34 °C. Cultures were passaged in 3 L BSK II media and allowed to reach a cell density of 3–5 × 107 cells · ml−1. Cells were collected by centrifugation at 10,000 × g for 30 min and then washed once in HN buffer  to remove residual BSK II. Cell pellets were resuspended in 20 ml of ice-cold lysis buffer containing 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 2 mM DTT, 5X protease inhibitor cocktail , and 200 U · ml−1 Turbonuclease . Cells were lysed by pushing the cell suspension through a 1-inch diameter French Pressure Cell twice at 3,000 PSI. Cell-free lysates were generated by removing insoluble cell debris from the lysed cell suspension with centrifugation at 4 °C, 20,000 × g for 30 min and then filtering the resulting supernatant through a 0.45 µm pore size syringe filter. His-tagged proteins were separated by nickel affinity chromatography. Lysates were loaded into a UPC-900 FPLC  and pumped through a HisTrapFF 1 mL  column. The column was washed with 9 mL of 5% elution buffer  and 6 mL 10% elution buffer. The resin-bound proteins were collected into elution fraction by increasing the gradient of elution buffer. The liquid volume of the RNA polymerase containing elution fractions were reduced by filtration in Amicon Ultra – 4  10 kDa pore size centrifugal filter columns. RNA polymerase was subjected to buffer exchange into a storage buffer containing 40 mM HEPES, 200 mM NaCl, and 2 mM DTT with a PD10 Sephadex G-25 column . The liquid volume was reduced to concentrate RNA polymerase on a centrifugal filter column, and final protein concentration was measured by spectrophotometry to determine A280nm without extinction coefficient adjustment (1 abs at 1 cm = 1 mg · ml−1) and BCA assay. The RNA polymerase was stored in 50% glycerol at −80 °C.The B. burgdorferi rpoD were commercially synthesized and cloned into the BamHI/EcoRI site  of the pMAL-C5X plasmid expression vector . The expression vector was transformed into Top Shot BL21 (DE3) pLysS Chemically Competent E. coli  to produce N-terminal Maltose binding protein (MBP)-tagged RpoD. Overnight E. coli cultures were passaged 1:200 into LB-Lennox broth containing 2 g · L−1 glucose and 100 µg · ml−1 ampicillin and then incubated at 32 °C until culture density reached optical density (OD 600 nm) of 0.5. The culture was incubated for an additional 2 hours with 0.3 mM isopropyl β-D-1-thiogalactopyranoside to allow protein expression under the lac operator. MBP-tagged proteins were purified from total cell extracts by amylose resin affinity chromatography as described in the pMAL-C5X protein expression system protocols . To remove the MBP-tag from the RpoD, 30 mg of purified protein was incubated over night with 200 µg Factor Xa protease in the presence of 2 mM calcium chloride. The mixture containing RpoD was separated by heparin affinity chromatography by flowing the mixture though a HiTrap Heparin HP column. Elution of the column with an increasing gradient of sodium chloride led to the release of recombinant RpoD to apparent homogeneity. RpoD was prepared for storage and use as described under the RNA polymerase purification section.Oligonucleotides encoding the codon-optimized version of B. burgdorferi B31 genome. Sequence matches were collated, and protein identities were ranked based on highest number of sequence matches normalized to predicted protein size.The purified RNA polymerase mixture was separated by SDS-PAGE. Protein bands stained with Imperial Protein Stain  were excised for analysis by LC/MS/MS at the Research Technology Branch, NIAID, NIH, . Following trypsin gel digest, protein samples were injected onto an Orbitrap Tribrid Mass Spectrometer equipped with Nano-LC Nano-Electrospray source . Data were analyzed with PEAKS v8.5  to discover sequences matching proteins encoded in the Borrelia RNA polymerase subunits β, α and σ70 (RpoD) . Linear detectable ranges of the polyclonal antibodies anti-RpoB, anti-RpoA, and anti-RpoD were determined by linear regression analysis. Analysis was performed on densitometry signals resulting from western blots loaded with twofold dilutions of recombinant target proteins and incubated with 1:2000 dilution of primary antibodies for 16 hours. Purified RNA polymerase was loaded in amounts to produce densitometry signals within the linear range (100–150 ng of purified protein) to quantify the RNA polymerase subunits purified by affinity chromatography. For western blotting, proteins were separated by SDS-PAGE in the Mini-Tetra Gel System  and transferred to PVDF membrane using the Transblot Turbo System . Primary antibodies were incubated with the membrane at a 1:2000 dilution for 17 hours. The antibodies bound to antigen were labeled by incubation of the membrane with HRP-conjugated protein-A  at a 1:4000 dilution for one hour and then the membrane was subjected to five 15-min washing steps in TBST. Labeled antibodies were detected by soaking the membrane with Super Signal West Pico chemiluminescent substrate kit  on the ChemiDoc MP imaging system . Chemiluminescent signals were quantified from ChemiDoc images by densitometry in Image Lab Software .Custom polyclonal antibodies were generated to full-length recombinant 75. Linear dsDNA templates were generated to detect sigma factor-dependent transcription from B. burgdorferi promoters. Transcriptional start sites upstream of genes of interest that were previously identified by either primer extension or RNAseq were targeted for template generation76. Primers were generated to amplify a 500-bp region that contains the previously annotated transcriptional start site in the middle of the amplicon .A single-stranded circular DNA template was generated for RNA polymerase activity (without sigma factor). A 45 bp oligonucleotide NC-45 (CTGGAGGAGATTTTGTGGTATCGATTCGTCTCTTAGAGGAAGCTA) was combined with splint oligonucleotide (CTCCAGTAGCTT) to a promote double-stranded complex and phosphodiester bond formation by T4 DNA ligase, resulting in single-stranded NC-45 oligonucleotide circularizationon Table . DNA wasin vitro transcription reactions were carried out under the following conditions unless otherwise stated. A 5X reaction buffer containing 300 mM potassium glutamate, 200 mM HEPES pH 7.5, 5 mM DTT, 0.25% NP40 detergent, 10 mM MgSO4, and 25 mM MnSO4 in HPLC grade water were stored for up to two weeks at −80 °C and mixed to a 1X concentration in the reaction mixture. The final reaction mixture contained 1X reaction buffer, 0.8 U RiboLock RNase inhibitor , 21 nM RNA polymerase, 500 nM RpoD, 2 µCi ATP [α-32P] , 20 µM ATP, 200 µM GTP, 200 µM CTP, and 200 µM UTP. A preliminary mixture containing reaction buffer, RiboLock RNase inhibitor , RNA polymerase, and RpoD was incubated for 10 min on ice prior to the addition of other components. Transcription was initiated with the addition of linear dsDNA template to a concentration of 10 nM, and reactions were allowed to proceed for 5 min at 37 °C. RNA products were separated by gel electrophoresis in 10% TBE-urea gels  at 180 V for 45 min. To detect accumulation of RNA that incorporated α-32P-ATP, gels were placed on a Phosphor Screen  overnight (16 hours), and the resulting signal was detected using the Typhoon FLA 9500 . Densitometry measurements were determined with Image Lab 6.0.1. Software .The Supplementary information.


Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection. The HIV-1 RNA-binding protein rev facilitates nuclear export of viral RNA. Here, the authors use native mass spectrometry to study the interactions between rev-derived peptides and rev response elements of HIV-1 RNA, providing mechanistic insights into rev recognition and recruitment. To gain insight into the biochemistry of retroviruses and further antiviral drug development, atomic-level details of RNA-protein complex structures and their binding interfaces can be studied by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. An emerging alternative that can complement these techniques, especially when RNA-protein complexes show unfavorable conformational dynamics that complicate NMR data interpretation3 or fail to crystallize properly5, is native top-down mass spectrometry (MS)6. We have recently demonstrated that native electrospray ionization (ESI)12 provides time-resolved, stoichiometric information on complexes of transactivation responsive (TAR) RNA and trans-activating (tat) peptide from human immunodeficiency virus type 1 (HIV-1), and that collisionally activated dissociation (CAD) can reveal tat binding sites of TAR RNA at the single-nucleotide level6. This native top-down MS approach relies on the preservation of electrostatic interactions between protonated peptides and deprotonated RNA at energies that are sufficiently high for phosphodiester backbone bond cleavage6, for which as few as two arginine residues are sufficient13.Interactions between proteins and ribonucleic acids (RNA) play important roles in biological processes including retroviral replication14, rev protein binding to rev response element (RRE) segments of unspliced and singly spliced viral mRNA facilitates their nuclear export16. Both tat and rev are thus critical to the expression of viral proteins and HIV-1 replication17. Previous studies have identified nucleotides in stem II of RRE RNA21 to which the arginine-rich motif (ARM) of rev protein binds19 in the assembly of the functional RRE/rev ribonucleoprotein complex in which up to 8–10 rev molecules sequentially associate with a single RRE molecule23. The N-terminal ARM binding region  of rev (116 residues) is framed by oligomerization domains  that stabilize the ribonucleoprotein complex15. However, as pointed out by Frankel, structural details on the formation of the ribonucleoprotein complex remain largely elusive, and one particularly important question is how the individual subunits of rev recognize the RRE4. Likewise, Le Grice stated that the sequence of events required for rev assembly and the positioning of individual rev molecules on the RRE remain unknown, and that the role of the three-way junction from which stem IIB originates in binding of the second rev molecule is unclear15. The as yet unresolved challenge to correlate binding regions with individual rev molecules that successively bind to RRE RNA complicates the rational design of highly efficient drugs for the treatment of HIV-1 infection.While tat binding to TAR RNA increases the production of viral messenger RNA (mRNA)RRE-IIB-0, RRE-TR-0, Fig. RRE-II-0) with rev ARM peptide24 are investigated by MS, and the data compared to results from NMR, X-ray crystallography, and biochemical probing experiments35. Our ESI and CAD data reveal a mechanism in which rev molecules are sequentially recruited by the upper stem of RRE IIB and relayed to binding sites that allow for rev dimerization rather than directly binding to individual sites as suggested by previous studies.Here we study the binding of an ARM peptide derived from rev protein to RRE RNA from HIV-1 by native ESI and CAD MS. Complexes of RRE stem IIB RNA constructs  were used in this study to limit the mass of the RRE/rev complexes, such that the signals of all fragments from CAD were isotopically resolved on our 7 T FT-ICR mass spectrometer for unambiguous assignment of fragment identity . Our MS data nevertheless provide meaningful insight into the assembly of the functional RRE/rev ribonucleoprotein complex as the interaction of rev protein with RRE RNA is modular, i.e., rev ARM peptide molecules bind to RRE RNA with the same affinity as rev protein monomer (rev protein with a mutation in its oligomerization domain to prevent rev oligomerization), and requires the same amino acids for recognition31. Furthermore, oligomerization-deficient rev protein was shown to assemble on RRE RNA36 one molecule at a time22 to produce complexes with ~12% nuclear export activity compared to those of wild-type rev protein36. In the RRE-IIB-0 RNA construct, A44•G76 of the wild-type RRE RNA was replaced by C44•G76, and two G•C pairs were added for stabilization of the lower stem. The same lower stem was used in the RRE-TR-0 construct, in which three base pairs  of the upper stem were removed and the wild-type loop was replaced by GCAA.The RRE RNA constructs R-0 Fig.  instead RRE-IIB-0 RNA (2 µM) and rev ARM peptide (4 µM) for ESI MS , shows signals of free RNA (~22%), 1:1 RNA-peptide complexes (~41%), and 1:2 RNA-peptide complexes (~37%). The 1:1 complexes must have formed at a relatively high rate as their yield was already ~32% at 5 min incubation time  was observed during the next ~8 h, and a yield of ~97% was only reached at 99 h incubation time  to RNA (2 µM) concentration ratio ([rev]0/[RRE]0) of 0.25 that limited overall complex formation to ~3% , on a time scale of up to 2700 s  reflects the formation of 1:1 complexes and the slow process (~0.0008 s−1) reflects the formation of 1:2 complexes, in agreement with the data in Fig. −1) that were ~17-fold higher than the rates for the formation of 1:2 complexes  = 100–7.5 ± 0.7·[rev]01.71 ± 0.08 , that extrapolates to 0% free RNA at [rev]0/[RRE]0 ~2.3. Likewise, the data in Fig. f([rev]0) = 0.086 ± 0.033·[rev]01.8 ± 0.3, that extrapolates to a ratio of 1:2 to 1:1 complex yields of ~2 at [rev]0/[RRE]0 = 3. We conclude that at least for incubation times between 1 and 5 h, 1:2 RRE/rev complexes constitute the major fraction of species in solutions of RRE and rev at concentrations that allow for binding of all RRE. Complexes of RNA constructs that resemble RRE stem IIB  to RRE RNA than both oligomerization-deficient rev protein (KD = 15 ± 1.7 nM) and rev ARM peptide (KD = 13 ± 2.0 nM).The data in Fig. RRE-IIB-0 RNA  values do not overlap. As shown in Fig. m/z values of (RNA + m·peptide - nH)n− ions with m = 0, 1, and 2 were well separated at all m and n values. For binding site mapping in the 1:1 and 1:2 complexes by CAD, we isolated (RRE-IIB-0 + 1·rev - 15H)15− ions at m/z ~1040 and (RRE-IIB-0 + 2·rev - 16H)16− ions at m/z ~1160, respectively. These ions were selected because of their high abundance 41 for RRE-IIB-0 RNA 14− ions at m/z ~1000 and (RRE-TR-0 + 2·rev - 14H)14− ions at m/z ~1214 (both ~0.4 charges/nt), respectively. CAD of the 1:1 complex produced c and y fragments with and without peptide attached 24.For binding site mapping in the 1:1 and 1:2 complexes by CAD, we isolated 13− ions—the abundance of (RRE-TR-0 + 1·rev - 14H)14− was too low for CAD at pH ~5.5—was virtually the same at pH ~5.5, ~7.5, and ~9.5 n− ions with n = 11–15 electrosprayed from a solution at pH ~7.5  whereas they do not bind to rev peptide in the NMR structure n− complexes at all n studied 14− ions, recorded ~6 min and ~155 min after preparation of a solution with RRE-TR-0 RNA (2 µM) and rev ARM peptide (4 µM), before and after the plateau region in Supplementary Fig. RRE-TR-0/rev 1:1 complexes did not differ for different incubation times and was very similar to that of the RRE-IIB-0/rev 1:1 complexes  and rev ARM peptide (1 and 2 µM) at pH ~8.1 (~10 min incubation time) showed free RNA and 1:1 and 1:5 RNA/peptide complexes  compared to that of y (~63%) fragments in the region 18–49  or G48-G50 (RRE-IIB-0) to compensate for the unavailability of A88-G91 in the stem IIB constructs. CAD of the 1:2 complexes of RRE-II-0  that can explain the observed slower binding of additional rev molecules  and the other of moderate (KD = 0.70 ± 0.29 μM) affinity31. Although the weaker binding event in the ITC study was interpreted as nonspecific binding31, our MS study shows that this binding event is specific  with ~400 µl aqueous ammonium acetate (100 mM) solution, followed by concentration to ~100 µl using centrifugal concentrators ; the concentration-dilution process was repeated five times and followed by six cycles of concentration and dilution with H2O. RRE-TR-0 RNA , RRE-IIB-0 RNA , and RRE-II-0 RNA  sequences were confirmed by CAD MS40. The rev ARM peptide  trifluoroacetate salts were custom synthesized by ThermoFisher Scientific, USA  and Sigma-Aldrich, Austria . Peptides were desalted by diluting ~200 µl rev peptide solution in H2O (~1 mM) with 1.8–3.8 ml aqueous ammonium acetate (100 mM) solution, followed by concentration to 200–500 µl using centrifugal concentrators ; the concentration-dilution process was repeated 4–5 times and followed by 5–6 cycles of concentration and dilution with H2O. The rev ARM peptide sequence was confirmed by electron capture dissociation (ECD) MS57. H2O was purified to 18 MΩ cm at room temperature using a Milli-Q system , CH3OH  was HPLC-grade, ammonium acetate (≥99.0%) and piperidine (≥99.5%) were from Sigma-Aldrich (Austria).RNA was prepared by solid phase synthesis−1 in negative ion mode; the potential difference between the ESI emitter (nebulizer) and the inlet capillary was 3100 or 3200 V and the desolvation gas (N2) temperature 180 °C. For native ESI, 1–1.25 mM piperidine58 was added to RNA/peptide solutions in 9:1 H2O/CH3OH (pH ~7.5 to ~8.1); our previous study on TAR/tat6 showed that the addition of 10% CH3OH at near-neutral pH did not alter the RNA/peptide binding region established by NMR spectroscopy. Further, data from chemical probing experiments with N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMCT) were, within error limits, the same for solutions with and without 10% CH3OH at pH ~8 .Prior to the addition of rev peptide solution, RNA solutions were heated to 90 °C for 90 s, followed by cooling on ice for ~10 min and thermal equilibration for another ~10 min at room temperature. Solutions of RNA (1–2 µM) and peptide (0.5–5 µM) were electrosprayed at a flow rate of 1.5 µl min59 (Argon gas flow through the collision cell was 0.2 l s−1), and an indirectly heated hollow cathode for electron capture dissociation (ECD)57. Electrostatic potentials and radiofrequency voltages of ion transfer elements  were optimized for maximum ion transmission, and the skimmer potential58 adjusted for efficient ion desolvation and dissociation of piperidine adducts . ESI spectra were obtained by operation of the linear quadrupole in transmission (radiofrequency-only) mode. For CAD, ions of interest were isolated in the quadrupole and dissociated in the collision cell using the laboratory frame collision energy indicated in the Figure legends 59. However, as discussed in detail in ref. 59, these collision energy values are only a relative measure of the energy available for dissociation. As a more general guideline, the laboratory-frame energy for CAD was adjusted for full sequence coverage and minimum secondary dissociation59 in each experiment. For ESI and CAD, 25–50 and 250–500 scans were added for each spectrum, respectively, and data reduction utilized the SNAP2 algorithm .All experiments were performed on a 7 T FT-ICR instrument  equipped with an ESI source, a linear quadrupole for ion isolation, a collision cell for CAD60. Only signals with a signal-to-noise ratio >3 and a mass accuracy <10 ppm were considered for analysis. Signals of rev ARM peptide monomers were observed in the ESI spectra in negative ion mode, but not included in the analysis as the ionization of compounds with high pI—that of rev ARM peptide is >12—in negative ion mode (wrong-way-round ESI) is far less efficient than in right-way-round ESI61. Likewise, the ionization efficiency of RNA in positive ion mode (wrong-way-round) is substantially reduced compared with ESI in negative ion mode (right-way-round)38. Occupancy values 6 of RRE RNA fragments with rev ARM peptide were calculated for each RNA cleavage site from site-specific yields (Y) of c and y fragments from CAD with 100% corresponding to the total number of rev molecules bound in each experiment, for CAD of 1:1 complexes as O = 100·Y(c or y with 1·rev)/(Y(c or y with 1·rev) + Y(c or y without rev)). For CAD of 1:2 complexes, occupancy values were calculated as O = 50·Y(c or y with 1·rev)/(Y(c or y with 1·rev) + Y(c or y without rev)) when Y(c or y with 2·rev) = 0, and as O = 50 + 50·Y(c or y with 2·rev)/(Y(c or y with 2·rev)+ Y(c or y with 1·rev)) when Y(c or y without rev) = 0. For CAD of 1:5 complexes, occupancy values were calculated accordingly such that the addition of each rev ARM peptide corresponds to an increase of 20%, i.e., O = 20·Y(c or y with 1·rev)/(Y(c or y with 1·rev) + Y(c or y without rev)) when Y(c or y with ≥2·rev) = 0, as O = 20 + 20·Y(c or y with 2·rev)/(Y(c or y with 2·rev)+ Y(c or y with 1·rev)) when Y(c or y with ≥3·rev) = 0, as O = 40 + 20·Y(c or y with 3·rev)/(Y(c or y with 3·rev) + Y(c or y with 2·rev)) when Y(c or y with ≥4·rev) = 0, as O = 60 + 20·Y(c or y with 4·rev)/(Y(c or y with 4·rev)+ Y(c or y with 3·rev)) when Y(c or y with ≥5·rev) = 0, and as O = 80 + 20·Y(c or y with 5·rev)/(Y(c or y with 5·rev)+ Y(c or y with 4·rev)) when Y(c or y with 5·rev) ≠ 0. Data from our previous study on TAR/tat6 showed that standard deviations of occupancy values varied between 0.1% and 7.9%, with average and median values of ~1.9% and 1.3%, respectively.Yields of free RNA, 1:1 complex, and 1:2 complex were calculated from the corresponding signal heigths in the spectra from ESI in negative ion mode, divided by the number of charges as ICR detector response is proportional to this number6, also listed in the Source Data file) by calculation of the standard deviation of residuals from exponential fit functions as 3σ = 6%. Errors in yield ratio (Fig. Errors for the yield data Fig. , 5 were tio Fig. , 5b, 5d Further information on research design is available in the Supplementary InformationReporting Summary


Immune checkpoint inhibitors (ICIs) are novel humanized monoclonal antibodies that release the brakes on the immune system, resulting in the destruction of tumor cells. ICIs are approved for a variety of hematological and solid organ malignancies, and the list has been growing since the approval of ipilimumab in 2011. ICIs are associated with a variety of immune-related adverse events (irAEs). irAEs commonly affect the skin, the gastrointestinal (GI) tract, and the endocrine system. Acute kidney injury (AKI) due to ICIs (ICI-AKI) occurs in a minority of patients, and it is usually due to acute tubulointerstitial nephritis (ATIN). Treatment with corticosteroids is usually successful. There have been reports of electrolyte disorders due to ICIs, including hyponatremia, hypocalcemia, hypokalemia, and Fanconi syndrome. The diagnosis of electrolyte disorders requires vigilance and routine laboratory monitoring. Immune checkpoint inhibitors (ICIs) represent a major advance in cancer treatment over the past decade. Immunotherapy has revolutionized cancer treatment and is playing an ever-increasing role in the management of many malignancies. The list of approved indications for ICIs is steadily increasing. ICIs are new arrows in the quiver alongside surgery, chemotherapy, and radiation therapy.T-cell activation requires two signals. The first signal is the interaction between T cell receptor (TCR) on T-lymphocytes and the major histocompatibility complex (MHC) on antigen-presenting cells (APC). The second signal is the interaction between B7 (CD 80/86) on APC and CD 28 on T-lymphocytes.CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) is a CD28 homolog on T-lymphocytes with a higher binding affinity for B7 (CD80/86) expressed on APC. CTLA-4 and B7 interaction inhibit T cell activation. CTLA-4 inhibitors, such as ipilimumab or tremelimumab, block this interaction and subsequently enhance T cell activation . ProgramIpilimumab, a monoclonal antibody that targets CTLA-4 and activates the immune system, was the first ICI approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of melanoma. Currently, there are seven ICIs on the market  used for oncology drugs divides the AEs associated with these medications into five grades: grade 1 (mild), grade 2 (moderate), grade 3 (severe but not immediately life-threatening), grade 4 (life-threatening), and grade 5 (death related to AE).In our discussion, we will use the AKI classification proposed by the Kidney Disease Improving Global Outcomes (KDIGO) Table  2]..2].Immune-related adverse events (irAEs) due to ICIsThe most common irAEs associated with the use of ICIs are related to the skin , the GI tract , the endocrine system , and the respiratory system  . Renal iThe focus of this review article is the renal adverse effects of ICIs, including AKI and electrolyte disorders. We present data from several meta-analyses and clinical studies. We also included data from case series and case reports deemed of clinical significance. FDA adverse events database on electrolyte disorders are reviewed as well.Incidence of ICI-AKIEarlier reports of ICI-AKI performed kidney biopsy to establish the underlying pathologic diagnosis. Kidney biopsies were done because the clinical entity was new and its characteristics were not well-defined. Later, the American Society of Clinical Oncology published guidelines discouraging kidney biopsy and favoring empirical glucocorticoid treatment when ICP-AKI is suspected . Later rManohar et al. conducted a meta-analysis involving 48 clinical trials that monitored renal function and serum electrolytes in a total of 11,482 patients while on treatment with pembrolizumab or nivolumab (both are PD-1 inhibitors) . The oveCortazar et al. analyzed data from phase 2 and 3 clinical trials involving 3,695 patients . The incMechanism, risk factors, and pathology of ICI-AKIThe mechanism of ICI-AKI is unclear. There are speculations that ICIs can elicit an autoimmune response against a potential antigen on the surface of renal tubular cells resulting in ATIN .The predominant lesion in patients who underwent a kidney biopsy to determine the etiology of ICI-AKI was ATIN ,7,9-11. 2), and using dual ICI therapy. The authors did not report on the complications of renal biopsies. Interestingly, concomitant use of PPIs has been shown to be a risk factor for ICI-AKI in other reports too [In the series reported by Cortazar et al., 138 patients had ICI-AKI, 43% of whom underwent a diagnostic kidney biopsy. In >90%, the pathology showed ATIN (20% of kidney biopsies had granulomatous ATIN) [orts too ,9-12, anThere are no distinguishing features between ATIN due to ICIs and ATIN due to other causes. Patients with ATIN due to ICIs may present with microhematuria, subnephrotic range proteinuria, sterile pyuria, and rarely eosinophilia ,9. In alA rare but potentially life-threatening adverse effect of ICIs is thrombotic microangiopathy. Few case reports have reported thrombotic thrombocytopenic purpura (TTP) in conjunction with ICIs immunotherapy. Recently Ali et al. reported a case of TTP in a patient treated with nivolumab and ipilimumab for metastatic renal cell carcinoma . To biopsy or not to biopsyThe role of kidney biopsy in the diagnosis of ICI-AKI remains to be defined . Some auManagement of ICI-AKIICIs should be stopped if KDIGO AKI stage 2 or 3 is diagnosed. Patients with KDIGO AKI stage 1 can be observed closely, and ICIs should be discontinued if AKI worsens. Corticosteroids are the treatment of choice for ICI-AKI, and they should be initiated in severe AKI cases as early as possible. If we combine the data from the above-referenced six studies ,7,9-12 wThe six studies included 215 patients with ICI-AKI, 82% (177 patients) were treated with corticosteroids, though many different regimens were reported. Some patients had received intravenous corticosteroids initially.Of the patients who received corticosteroids, 79% had a partial or complete recovery. Some patients died of their primary malignancy while on corticosteroids.A total of 52 patients were rechallenged with ICIs (some while still on corticosteroids), of whom 18 patients (35%) had a recurrence of ICI-AKI.In the study by Cortazar et al., 31 patients were re-challenged, seven (23%) of whom developed recurrent ICI-AKI. Of the seven patients, only one did not recover from ICI-AKI . At the When patients were treated with oral corticosteroids, a common practice was to start prednisone at 1 mg/kg and to taper it slowly over eight to 12 weeks.Corticosteroids are the mainstay of treatment. Other immunosuppressive agents used were mycophenolate mofetil, rituximab, cyclophosphamide, and infliximab ,11.ICI use in kidney transplant and end-stage kidney disease (ESKD)Venkatachalam et al. reported poor renal outcomes in kidney transplant patients treated with ICIs . Their rThere is a paucity of data on the use of ICIs in patients with ESKD. Hirsch et al. reported eight patients with ESKD  who were treated with ICIs . There wThe characteristics of ICI-AKI are summarized in Table Electrolyte disorders due to ICIsElectrolyte disorders are important adverse reactions of ICIs. Examples include hyponatremia, hypocalcemia, hypercalcemia, hypomagnesemia, hypokalemia, hyperkalemia, hypophosphatemia, and Fanconi syndrome.The meta-analysis by Manohar et al. included five randomized controlled trials (RCTs) that assessed the association between PD-1 inhibitors and electrolyte disorders . In patiWanchoo et al. reviewed the FDA adverse events database from the third quarter of 2011 until the first quarter of 2015 . They idThere have been several case reports of severe electrolyte abnormalities associated with the use of ICIs. Win et al. reported a case of primary hypoparathyroidism in a 73-year old man with metastatic melanoma who presented to the emergency department with severe symptomatic hypocalcemia  . He had Balakrishna et al. reported the case of a 58-year-old man with a history of metastatic melanoma who presented with progressive quadriplegia after 10 months of treatment with nivolumab . InitialThe authors of this review have reported the first case of Fanconi syndrome associated with the use of ICIs (published online first in September 2019) . The patFarid et al. published a second case report of Fanconi syndrome due to ICIs . The patAKI is a rare complication of checkpoint inhibitor immunotherapy. The discontinuation of ICIs and treatment with corticosteroids are indicated for patients with severe renal injury but not in all cases. The most common reported pathology is ATIN, but immune complex glomerulonephritis and thrombotic microangiopathy (TMA) have also been observed. ICI-AKI can occur weeks or months after the initiation of treatment with ICIs. Some patients can be cautiously rechallenged with ICIs. A kidney biopsy is required for the diagnosis if an alternative diagnosis, such as acute glomerulonephritis or acute tubular injury, is suspected. Importantly, some patients develop a variety of electrolyte disorders such as hypocalcemia, hyponatremia, hypokalemia, and Fanconi syndrome. These electrolyte disorders can be severe and require regular laboratory monitoring.


Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P  and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss  REMSA was performed according to the method of Goto et al.  variant has the first PPR motif with [NN] instead of [VN]  and transformed the mutated PpPPR_56 into Δ of [VN] , while tn 56comp . Furtheromp line .nad3 sequence in 1L4L7L, and two C-terminal L motifs were modified in 10L13L   . TherefoThe contribution of the P and S motifs to RNA recognition in PLS-class PPR proteins has been experimentally confirmed , 16. Howin vitro binding studies after expression in E. coli and purification of recombinant proteins. However, we were unable to purify full-length PpPPR_56 fused to a His-tag in C-terminus, although it was detected in the soluble fraction by immunoblot analysis using anti-His-tag antibody. The structure of the C-terminus of the DYW domain is yet unknown, but it is possible that the folding of the C-terminal region could hide the His-tag and prevent the purification of the full-length PpPPR_56 with Ni-NTA agarose beads. A recombinant PpPPR_56 lacking the DYW domain was designed to bypass this problem. However, it has been recently shown that the DYW domain of CRR2 influences the specific interaction between the E domain and its target RNA [To determine whether the L motif play a role in the RNA recognition, we first performed rget RNA . Therefoet al. [et al [nad4. Here, we showed that 56ΔDYW-nad3L, a PpPPR_56 variant with completely altered L motifs to recognize the nad3 sequence, exhibited no significant difference from 56ΔDYW-wt in terms of nad3 RNA binding activity  in order to increase the RNA binding activity of the PPR protein . MORF/RI E. coli . Howeveret al. [PPR proteins are the potential candidates for RNA manipulation tools because each PPR motif recognizes one nucleotide, based on a simple code comprising two or three amino acids . This meet al.  successfS1 Fig32P-labeled psbI RNA (PpPsbI-RNA1 [(A) Schematic diagrams of the recombinant 56ΔDYW-wt and its variants . Amino acids at positions 5 and last of each PPR motif are indicated. Altered amino acid residues are shown in red. Trx: thioredoxin; V5: V5 epitope tag; and His: 6× histidine tag. (B) Equal amounts (1 μg) of r56ΔDYW proteins  were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining. Black arrowhead indicates the predicted size of the 56ΔDYW proteins. (C) REMSA was performed with 56ΔDYW proteins , and sbI-RNA1 ). The co(EPS)Click here for additional data file.S2 Fignad3 and nad4 RNAs. Key amino acids at positions 5 and last of each PPR motif are indicated. Altered amino acid residues are shown in red. Aligned nucleotides are shaded in gray to indicate matches to the proposed RNA recognition codes. (B) RNA editing levels in PpPPR_56 KO mosses complemented with variants. The cDNA sequencing chromatograms and RNA editing efficiencies (%) are shown. Black arrowheads indicate editing sites.(A) Alignment of PpPPR_56 and its variants to (EPS)Click here for additional data file.S3 Fig32P-labeled RNAs (nad3 and nad4). B and U indicate bound and unbound probe, respectively. (B) The histogram shows the percentage of probes (nad3 and nad4) shifted with 50 nM of 56ΔDYW-wt and its variants . Error bars indicate ± SD (n = 3).(A) REMSA was performed using 50 nM of 56ΔDYW proteins  and (EPS)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Offset schemes help avoid or revert habitat loss through protection of existing habitat (avoided deforestation), through the restoration of degraded areas , or both. The spatial scale of an offset scheme may influence which of these 2 outcomes is favored and is an important aspect of the scheme's design. However, how spatial scale influences the trade‐offs between the preservation of existing habitat and restoration of degraded areas is poorly understood. We used the largest forest offset scheme in the world, which is part of the Brazilian Forest Code, to explore how implementation at different spatial scales may affect the outcome in terms of the area of avoided deforestation and area of regrowth. We employed a numerical simulation of trade between buyers  and sellers  in the Brazilian Amazon to estimate potential avoided deforestation and regrowth at different spatial scales of implementation. Allowing offsets over large spatial scales led to an area of avoided deforestation 12 times greater than regrowth, whereas restricting offsets to small spatial scales led to an area of regrowth twice as large as avoided deforestation. The greatest total area (avoided deforestation and regrowth combined) was conserved when the spatial scale of the scheme was small, especially in locations that were highly deforested. To maximize conservation gains from avoided deforestation and regrowth, the design of the Brazilian forest‐offset scheme should focus on restricting the spatial scale in which offsets occur. Such a strategy could help ensure conservation benefits are localized and promote the recovery of degraded areas in the most threatened forest landscapes. Article impact statement: Conservation offset schemes may yield greater additionality through avoided deforestation and restoration if implemented in smaller areas. A variety of mechanisms have been developed to manage human‐caused habitat change and promote outcomes that aid conservation  . The FCWe focused on the Amazon, the world's largest standing forest, which covers 400 million hectares  inside a PA that is at least equivalent to their LR deficit and donate to the environmental agency (option 1). This option allows for a perpetual solution for buyers, which appears to be their preferred option  . The CRA is a hectare‐by‐hectare market, wherein a buyer can lease the extra LRs of several sellers or one seller can supply several buyers (option 2). Instead of a single perpetual transaction as the acquisition of land in PAs, CRA consists of contracts in which the price of the hectare and the duration of the contract are decided between buyers and sellers. Some buyers may prefer to offset their LR deficit by allowing the natural regrowth of secondary forest (option 3) . Offsets occurred in all PAs, CRA, and regrowth (scenario 1). Offsets were allowed on private land inside all PAs. The text in the FC is not specific about whether the PA must be a federal, state, or municipal area. Thus, this scenario included offsets in all PAs first, followed by CRA offset trade, and then the remaining buyers were assigned to offset through regrowth.Offsets occurred in federal PAs, CRA, and regrowth (scenario 2). The Ministry of the Environment established a regulatory framework Brasil  that conOffsets occurred with CRA and regrowth (scenario 3). This scenario ruled out offsets on private land inside any PA and included only CRA trade first, and then remaining buyers were assigned to offset through regrowth.Scenario 1 included all offset options stated by law—the most permissive scenario. Scenarios 2 and 3 gradually imposed policy restrictions associated with the offsetting options. The different policy scenarios were established to examine whether the results of our simulations would be driven by the spatial scales or by the different rules of the policy scenarios. For example, offset in PAs is unique to the FC, and removing this option allowed a more general examination of the effect of scale on offset schemes outcomes.We assumed compliance is a buyer‐led strategy because noncompliance incurs severe penalties, such as fines, land embargoes, or denial of access to loans. Therefore, buyers actively sought sellers in our analysis. Offset in PAs appears to be preferred by buyers because, besides being in perpetuity , we developed an algorithm (Supporting Information) in which each buyer sought the best‐matching seller. The algorithm is deterministic, meaning the deals between buyers and sellers are established based on the smallest difference between buyer's deficit and seller's surplus. In the case of offsetting in PAs, buyers must acquire an area that is at least equivalent to their deficit; thus, only one transaction was allowed. We assumed that, given their low cost, buyers would be willing to purchase an area that was up to 20% larger than their LR deficit. If such conditions were met, a buyer was considered compliant. If not, a buyer remained noncompliant and available for CRA trade.The CRA is a hectare‐by‐hectare offset market; thus, buyers looked for sellers that had the most similar area of native vegetation surplus to their deficit. Buyers were considered compliant if they managed to offset all their deficit in 3 transactions . We assumed a limited number of transactions because the general behavior of landowners is to minimize transaction costs associated with each trade  in which we progressively increased the 20% limit of private land inside PAs and increased the number of transactions allowed in CRA to assess whether our results are robust.The best‐match algorithm was iterated for each of the 3 policy scenarios at each of the 5 spatial scales. After each of the 15 simulations, we computed the sum of total offset (in Mha) for each of the 3 compliance options: offset in PAs, CRA, and regrowth. Offset in PAs was divided into 2 conservation outcomes: the area of potential regrowth and the area representing nonadditional offset. Private land in PAs, after acquired by buyers, must be donated to the statutory environmental agency, making their cleared portions likely to be allocated to regrowth. The area covered by natural vegetation was therefore considered nonadditional because it is already protected. Total offset with CRA was also divided into 2 conservation outcomes: avoided deforestation and nonadditional offset. Avoided deforestation corresponded to offsets occurring in unprotected native vegetation . Last, offsets through regrowth outside PAs were computed as a single conservation outcome.To calculate total additionality for each simulation, we summed avoided deforestation and regrowth (inside and outside PAs), assuming these are both conservation benefits that would not occur in the absence of the offset scheme. Nonadditional outcomes represented offsets in already protected vegetation that are mostly in the land of smallholders and settlements. Thus, the key conservation outcomes of this analysis were avoided deforestation, regrowth, and total additionality. Finally, for each policy scenario and spatial scale, we calculated the percentages of each conservation outcome based on the total deficit to assess the proportion of total forest deficit that was effectively converted to a conservation gain.The total native vegetation deficit across the Brazilian Amazon was 4.94 Mha, whereas the total supply of native vegetation that could be used for compensation was 10 times greater (50 Mha) . This result indicated that even if our best‐match assumptions were more flexible, our findings would likely remain the same, and that smaller scales would still result in more additionality than larger scales.In all scenarios, across all spatial scales, most of the offset was nonadditional. In scenarios 1 and 2, offsets in PAs generated little regrowth compared to the total offset. For CRA offsets, avoided deforestation was lower than the nonadditional offsets. Overall, the total additionality was <50% in all scenarios; scenario 3 resulted in the greatest total additionality across all scales , land‐tenure data preparation (S2), land‐cover data (S3), classification of buyers and sellers (S4), matching algorithm (S5), and sensitivity analysis (S6) are available online. The authors are solely responsible for the content and functionality of these materials. Queries  should be directed to the corresponding author.Click here for additional data file.   Click here for additional data file.