Polyamine metabolism controls B-to-Z DNA transition to orchestrate DNA sensor cGAS activity.

Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.

DNA triplet repeat expansion and mismatch repair.

DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSß and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.

S1-END-seq reveals DNA secondary structures in human cells.

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Enriched G4 forming repeats in the human genome are associated with robust well-coordinated transcription and reduced cancer transcriptome variation.

Non-B DNA G-quadruplex (G4) structures with guanine (G) runs of 2 to 4 repeats can trigger opposing experimental transcriptional impacts. Here, we used bioinformatic algorithms to comprehensively assess correlations of steady-state RNA transcript levels with all putative G4 sequence (pG4) locations genome-wide in three mammalian genomes and in normal and tumor human tissues. The human pG4-containing gene set displays higher expression levels than the set without pG4, supporting and extending some prior observations. pG4 enrichment at transcription start sites (TSSs) in human, but not chimpanzee and mouse genomes, suggests possible positive selection pressure for pG4 at human TSS, potentially driving genome rewiring and gene expression divergence between human and chimpanzee. Comprehensive bioinformatic analyses revealed lower pG4-containing gene set variability in humans and among different pG4 genes in tumors. As G4 stabilizers are under therapeutic consideration for cancer and pathogens, such distinctions between human normal and tumor G4s along with other species merit attention. Furthermore, in germline and cancer sequences, the most mutagenic pG4 mapped to regions promoting alternative DNA structures. Overall findings establish high pG4 at TSS as a human genome attribute statistically associated with robust well-coordinated transcription and reduced cancer transcriptome variation with implications for biology, model organisms, and medicine.

Structural morphing in the viral portal vertex of bacteriophage lambda.

The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system. IMPORTANCE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones.

DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress. This damage causes DNA lesions that, if not repaired quickly, are prone to nuclease cleavage, resulting in DSBs. Our results provide mechanistic insight into why human neoplastic translocation fragile DNA sequences are more prone to enzymes or agents that cause longer-lived DNA lesions.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Triple-helix potential of the mouse genome.

Certain DNA sequences, including mirror-symmetric polypyrimidine•polypurine runs, are capable of folding into a triple-helix­containing non­B-form DNA structure called H-DNA. Such H-DNA­forming sequences occur frequently in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)­specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides an approach for studying DNA secondary structure genome-wide at high spatial resolution.

The mechanisms of human lymphoid chromosomal translocations and their medical relevance.

The most common human lymphoid chromosomal translocations involve concurrent failures of the recombination activating gene (RAG) complex and Activation-Induced Deaminase (AID). These are two enzymes that are normally expressed for purposes of the two site-specific DNA recombination processes: V(D)J recombination and class switch recombination (CSR). First, though it is rare, a low level of expression of AID can introduce long-lived T:G mismatch lesions at 20-600 bp fragile zones. Second, the V(D)J recombination process can occasionally fail to rejoin coding ends, and this failure may permit an opportunity for Artemis:DNA-dependent kinase catalytic subunit (DNA-PKcs) to convert the T:G mismatch sites at the fragile zones into double-strand breaks. The 20-600 bp fragile zones must be, at least transiently, in a single-stranded DNA (ssDNA) state for the first step to occur, because AID only acts on ssDNA. Here we discuss the key DNA sequence features that lead to AID action at a fragile zone, which are (a) the proximity and density of strings of cytosine nucleotides (C-strings) that cause a B/A-intermediate DNA conformation; (b) overlapping AID hotspots that contain a methyl CpG (WRCG), which AID converts to a long-lived T:G mismatch; and (c) transcription, which, though not essential, favors increased ssDNA in the fragile zone. We also summarize chromosomal features of the focal fragile zones in lymphoid malignancies and discuss the clinical relevance of understanding the translocation mechanisms. Many of the key principles covered here are also relevant to chromosomal translocations in non-lymphoid somatic cells as well.

Young retrotransposons and non-B DNA structures promote the establishment of dominant rye centromere in the 1RS.1BL fused centromere.

The fine centromere structure in Robertsonian wheat-rye translocation chromosomes exhibits variation among different translocation genotypes. Within extensively employed wheat-rye 1RS.1BL translocation lines in wheat breeding, their translocated chromosomes frequently display fused centromere. Nevertheless, the mechanism governing the functionality of the fused centromere in 1RS.1BL translocated chromosomes remains to be clarified. In this study, we investigated the fine centromere structure of the 1RS.1BL translocated chromosome through a combination of cytological and genomics methods. We found that only the rye-derived centromere exhibits functional activity, whether in breeding applications or artificially synthesized translocation chromosomes. The active rye-derived centromere had higher proportion of young full-length long terminal repeat retrotransposons (flLTR-RTs) and more stable non-B DNA structures, which may be beneficial toward transcription of centromeric repeats and CENH3 loading to maintain the activity of rye centromeres. High levels of DNA methylation and H3K9me2 were found in the inactive wheat-derived centromeres, suggesting that it may play a crucial role in maintaining the inactive status of the wheat centromere. Our works elucidate the fine structure of 1RS.1BL translocations and the potential mechanism of centromere inactivation in the fused centromere, contributing knowledge to the application of fused centromere in wheat breeding formation of new wheat-rye translocation lines.

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

The Binding Properties of Antibodies to Z-DNA in the Sera of Normal Healthy Subjects.

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin.

The expression of antibodies to Z-DNA in the blood of patients with systemic lupus erythematosus: Relationship to autoantibodies to B-DNA.

To explore the antibody response to Z-DNA, a DNA conformation with a zig-zag structure, blood of patients with systemic lupus erythematosus (SLE) and otherwise healthy individuals (NHS) were assayed by ELISA using brominated poly(dGdC), a synthetic Z-DNA antigen. These studies showed that SLE patients commonly express antibodies to Z-DNA; NHS also had binding in this assay. In SLE blood, levels of antibodies to Z-DNA were related to those to B-DNA using calf thymus DNA as a source of B-DNA; cross-reactivity was demonstrated by adsorption experiments using DNA cellulose. As shown by dissociation assays, antibody binding of SLE anti-Z-DNA is sensitive to the effects of ionic strength, suggesting electrostatic binding. Since Z-DNA structure can be found in bacterial DNA as well as bacterial biofilms, these findings suggest that, in SLE, anti-DNA antibody responses can result from stimulation by DNA of bacterial origin, with cross-reactivity leading to autoreactivity.

DNAgenie: accurate prediction of DNA-type-specific binding residues in protein sequences.

Efforts to elucidate protein-DNA interactions at the molecular level rely in part on accurate predictions of DNA-binding residues in protein sequences. While there are over a dozen computational predictors of the DNA-binding residues, they are DNA-type agnostic and significantly cross-predict residues that interact with other ligands as DNA binding. We leverage a custom-designed machine learning architecture to introduce DNAgenie, first-of-its-kind predictor of residues that interact with A-DNA, B-DNA and single-stranded DNA. DNAgenie uses a comprehensive physiochemical profile extracted from an input protein sequence and implements a two-step refinement process to provide accurate predictions and to minimize the cross-predictions. Comparative tests on an independent test dataset demonstrate that DNAgenie outperforms the current methods that we adapt to predict residue-level interactions with the three DNA types. Further analysis finds that the use of the second (refinement) step leads to a substantial reduction in the cross predictions. Empirical tests show that DNAgenie's outputs that are converted to coarse-grained protein-level predictions compare favorably against recent tools that predict which DNA-binding proteins interact with double-stranded versus single-stranded DNAs. Moreover, predictions from the sequences of the whole human proteome reveal that the results produced by DNAgenie substantially overlap with the known DNA-binding proteins while also including promising leads for several hundred previously unknown putative DNA binders. These results suggest that DNAgenie is a valuable tool for the sequence-based characterization of protein functions. The DNAgenie's webserver is available at http://biomine.cs.vcu.edu/servers/DNAgenie/.

Synchrotron radiation circular dichroism spectroscopy of oligonucleotides at millimolar concentrations.

Circular dichroism spectroscopy of nucleic acids has been traditionally performed at sample concentrations orders of magnitude lower than what occur in biological systems. While recent work from us demonstrated the flexibility of an adjustable sample cell that allowed for successful recording of CD spectra of an 18- and a 21-mer double stranded DNA sequences at around 1 mM, sample concentrations beyond 1 mM present a challenge for standard benchtop CD spectrometers. In the present work, the synchrotron radiation circular dichroism (SRCD) spectra were recorded for d(CG)9 and a mixed 18-mer double stranded DNA at 1, 5, and 10 mM in 100 mM or 4 M NaCl. SRCD of low molecular weight salmon DNA was also measured at a 10 mg/ml concentration. These results represent the first report of CD spectra of DNA samples measured at concentrations comparable to those found in the nucleus. The results suggest that dsDNA maintain very similar structures at concentrations up to tens of mg/ml, as evident by the very similar CD patterns in this concentration range. Furthermore, the SRCD allowed for the recording of CD patterns of DNA in the far UV region, which is not readily accessible by standard benchtop CD spectropolarimeters. These far UV signals appear to be quite characteristic of DNA structures and are sensitive to sample conditions.

Circular dichroism and UV-Vis detection of UV-induced damage to nucleic acids.

In this report, we demonstrate that CD spectroscopy can be used as a tool to detect changes to DNA upon irradiation with UV light. We follow the spectroscopic response of DNA samples irradiated at selected exposure times with both CD and UV-Vis spectroscopy. We analyzed four different nucleic acids to evaluate the effect of the sequence on photodegradation. Only one polymer, calf thymus DNA, was a natural nucleic acid and contained all four nucleobases. The other three were synthetic polymers and contained only one type of base pair: poly (deoxyadenylic-deoxythymidylic) acid [poly (dA-dT)2 ] and poly (deoxyadenylic acid) · poly (deoxythymidylic acid) [poly (dA) · poly (dT)], which contained only adenine and thymine; poly (deoxyguanylic-deoxycytidylic) acid [poly (dG-dC)2 ], which contained only guanine and cytosine. CD and UV-Vis spectra showed sequence dependent changes. In particular, poly (dA) · poly (dT) undergoes changes more rapidly than the other sequences investigated. The CD spectrum of poly (dA) · poly (dT) gradually undergoes an inversion, suggesting a change in helicity, before disappearing due to the unfolding of the double strand.

Distinct sequence features underlie microdeletions and gross deletions in the human genome.

Microdeletions and gross deletions are important causes (~20%) of human inherited disease and their genomic locations are strongly influenced by the local DNA sequence environment. This notwithstanding, no study has systematically examined their underlying generative mechanisms. Here, we obtained 42,098 pathogenic microdeletions and gross deletions from the Human Gene Mutation Database (HGMD) that together form a continuum of germline deletions ranging in size from 1 to 28,394,429 bp. We analyzed the DNA sequence within 1 kb of the breakpoint junctions and found that the frequencies of non-B DNA-forming repeats, GC-content, and the presence of seven of 78 specific sequence motifs in the vicinity of pathogenic deletions correlated with deletion length for deletions of length ≤30 bp. Further, we found that the presence of DR, GQ, and STR repeats is important for the formation of longer deletions (>30 bp) but not for the formation of shorter deletions (≤30 bp) while significantly (χ2 , p < 2E-16) more microhomologies were identified flanking short deletions than long deletions (length >30 bp). We provide evidence to support a functional distinction between microdeletions and gross deletions. Finally, we propose that a deletion length cut-off of 25-30 bp may serve as an objective means to functionally distinguish microdeletions from gross deletions.

Context-dependent DNA polymerization effects can masquerade as DNA modification signals.

BACKGROUND: Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD). RESULTS: In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples. CONCLUSION: The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.

Evaluation of dried blood spots for hepatitis B and D serology and nucleic acid testing.

Areas with the highest burden of hepatitis B virus (HBV) infection are often low-middle-income countries with limited access to diagnosis due to isolation, affordability, and/or feasibility. Dried blood spots (DBSs) provide an alternative for remote areas where collection and transportation of serum is impractical. In this study, the application of DBS for serological and molecular detection of HBV and hepatitis D virus (HDV) was evaluated. Hepatitis B surface antigen was detected in 87 of 91 (95.6%) DBS. Seventeen of 21 (81%) had detectable HBeAg and 52 of 71 (73.2%) were anti-HBe positive. Anti-HD was detectable in 11 of 12 (91.6%) spiked control DBS after an initial failure to detect in patient DBS. HBV DNA was detected from 50 of 70 (71.4%) DBS with serum loads greater than 200 IU/mL in an in-house assay and 18 of 24 (75%) DBS with loads exceeding 389 IU/mL in a commercial assay. Using linear regression, HBV DNA loads from DBS were able to predict serum loads in 46 of 50 (92%) samples to within 1 log of actual serum load. HDV RNA was detected in 42 of 47 (89%) DBS with serum levels greater than 7200 IU/mL. DBSs are recommended for diagnosis of HBV, monitoring, and detection of high loads in pregnant women where peripheral blood testing remains unfeasible. Detection of HDV RNA from DBS may prove useful in endemic areas.