Sequence dependent phase separation of protein-polynucleotide mixtures elucidated using molecular simulations.

Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid-liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.

Enhanced Prodigiosin Production in Serratia marcescens JNB5-1 by Introduction of a Polynucleotide Fragment into the pigN 3' Untranslated Region and Disulfide Bonds into O-Methyl Transferase (PigF).

In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.

Prebiotic competition and evolution in self-replicating polynucleotides can explain the properties of DNA/RNA in modern living systems.

BACKGROUND: We hypothesize prebiotic evolution of self-replicating macro-molecules (Alberts, Molecular biology of the cell, 2015; Orgel, Crit Rev Biochem Mol Biol 39:99-123, 2004; Hud, Nat Commun 9:5171) favoured the constituent nucleotides and biophysical properties observed in the RNA and DNA of modern organisms. Assumed initial conditions are a shallow tide pool, containing a racemic mix of diverse nucleotide monomers (Barks et al., Chembiochem 11:1240-1243, 2010; Krishnamurthy, Nat Commun 9:5175, 2018; Hirao, Curr Opin Chem Biol 10:622-627), subject to day/night thermal fluctuations (Piccirilli et al., Nature 343:33-37, 1990). Self-replication, like Polymerase Chain Reactions, followed as higher daytime thermal energy "melted" inter-strand hydrogen bonds causing strand separation while solar UV radiation increased prebiotic nucleobase formation (Szathmary, Proc Biol Sci 245:91-99, 1991; Materese et al., Astrobiology 17:761-770, 2017; Bera et al., Astrobiology 17:771-785, 2017). Lower night energies allowed free monomers to form hydrogen bonds with their template counterparts leading to daughter strand synthesis (Hirao, Biotechniques 40:711, 2006). RESULTS: Evolutionary selection favoured increasing strand length to maximize auto-catalytic function in RNA and polymer stability in double stranded DNA (Krishnamurthy, Chemistry 24:16708-16715, 2018; Szathmary, Nat Rev Genet 4:995-1001, 2003). However, synthesis of the full daughter strand before daytime temperatures produced strand separation, longer polymer length required increased speed of self-replication. Computer simulations demonstrate optimal polynucleotide autocatalytic speed is achieved when the constituent nucleotides possess a left-right asymmetry that decreases the hydrogen bond kinetic barrier for the free nucleotide attachment to the template on one side and increases bond barrier on the other side preventing it from releasing prior to covalent bond formation. This phenomenon is similar to asymmetric kinetics observed during polymerization of the front and the back ends of linear cytoskeletal proteins such as actin and microtubules (Orgel, Nature 343:18-20, 1990; Henry, Curr Opin Chem Biol 7:727-733, 2003; Walker et al., J Cell Biol 108:931-937, 1989; Crevenna et al., J Biol Chem 288:12102-12113, 2013). Since rotation of the nucleotide would disrupt the asymmetry, the optimal nucleotides must form two or more hydrogen bonds with their counterpart on the template strand. All nucleotides in modern RNA and DNA have these predicted properties. Our models demonstrate these constraints on the properties of constituent monomers result in biophysical properties found in modern DNA and RNA including strand directionality, anti-parallel strand orientation, homochirality, quadruplet alphabet, and complementary base pairing. Furthermore, competition between RNA and DNA auto-replicators for 3 nucleotides in common permit states coexistence and possible cooperative interactions that could be incorporated into nascent living systems. CONCLUSION: Our findings demonstrate the molecular properties of DNA/RNA could have emerged from Darwinian competition among macromolecular replicators that selected nucleotide monomers that maximized the speed of autocatalysis.

KnotGenome: a server to analyze entanglements of chromosomes.

The KnotGenome server enables the topological analysis of chromosome model data using three-dimensional coordinate files of chromosomes as input. In particular, it detects prime and composite knots in single chromosomes, and links between chromosomes. The knotting complexity of the chromosome is presented in the form of a matrix diagram that reveals the knot type of the entire polynucleotide chain and of each of its subchains. Links are determined by means of the Gaussian linking integral and the HOMFLY-PT polynomial. Entangled chromosomes are presented graphically in an intuitive way. It is also possible to relax structure with short molecular dynamics runs before the analysis. KnotGenome is freely available at http://knotgenom.cent.uw.edu.pl/.

Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c7dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide.

Structurally Distinct Cation Channelrhodopsins from Cryptophyte Algae.

Microbial rhodopsins are remarkable for the diversity of their functional mechanisms based on the same protein scaffold. A class of rhodopsins from cryptophyte algae show close sequence homology with haloarchaeal rhodopsin proton pumps rather than with previously known channelrhodopsins from chlorophyte (green) algae. In particular, both aspartate residues that occupy the positions of the chromophore Schiff base proton acceptor and donor, a hallmark of rhodopsin proton pumps, are conserved in these cryptophyte proteins. We expressed the corresponding polynucleotides in human embryonic kidney (HEK293) cells and studied electrogenic properties of the encoded proteins with whole-cell patch-clamp recording. Despite their lack of residues characteristic of the chlorophyte cation channels, these proteins are cation-conducting channelrhodopsins that carry out light-gated passive transport of Na(+) and H(+). These findings show that channel function in rhodopsins has evolved via multiple routes.

Length heteroplasmy of the polyC-polyT-polyC stretch in the dog mtDNA control region.

Previously, the mitochondrial control region of 214 Belgian dogs was sequenced. Analysis of this data indicated length heteroplasmy of the polyT stretch in the polyC-polyT-polyC stretch from positions 16661 to 16674. Nine polyC-polyT-polyC haplotype combinations were observed, consisting of seven major haplotypes (highest signal intensity) combined with minor haplotypes (lower signal intensity) one T shorter than the major haplotype in all but three dogs. The longer the polyT stretch, the smaller was the difference in signal intensity between the major and minor haplotype peaks. Additional sequencing, cloning, and PCR trap experiments were performed to further study the intra-individual variation of this mitochondrial DNA (mtDNA) region. Cloning experiments demonstrated that the proportion of clones displaying the minor haplotypes also increased with the length of the polyT stretch. Clone amplification showed that in vitro polymerase errors might contribute to the length heteroplasmy of polyT stretches with at least 10 Ts. Although major and minor polyC-polyT-polyC haplotypes did not differ intra-individually within and between tissues in this study, interpretation of polyT stretch variation should be handled with care in forensic casework.

[The role of Ku antigen in the repair of apurinic/apyrimidinic sites in DNA].

Apurinic/apyrimidinic (AP) sites are some of the most frequent lesions in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku)--the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair--was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here, Ku roles in NHEJ and BER pathways of DNA repair are overviewed.

The Mycoplasma gallisepticum virulence factor lipoprotein MslA is a novel polynucleotide binding protein.

Although lipoproteins of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few have had their biochemical function defined. The gene encoding the lipoprotein MslA in Mycoplasma gallisepticum has recently been shown to be required for virulence, but the biochemical function of this gene is not known. Although this gene has no significant sequence similarity to any gene of known function, it is located within an operon in M. gallisepticum that contains a homolog of a gene previously shown to be a nonspecific exonuclease. We mutagenized both genes to facilitate expression in Escherichia coli and then examined the functions of the recombinant proteins. The capacity of MslA to bind polynucleotides was examined, and we found that the protein bound single- and double-stranded DNA, as well as single-stranded RNA, with a predicted binding site of greater than 1 nucleotide but less than or equal to 5 nucleotides in length. Recombinant MslA cleaved into two fragments in vitro, both of which were able to bind oligonucleotides. These findings suggest that the role of MslA may be to act in concert with the lipoprotein nuclease to generate nucleotides for transport into the mycoplasma cell, as the remaining genes in the operon are predicted to encode an ABC transporter.

Virus replication as a phenotypic version of polynucleotide evolution.

In this paper, we revisit and adapt to viral evolution an approach based on the theory of branching process advanced by Demetrius et al. (Bull. Math. Biol. 46:239-262, 1985), in their study of polynucleotide evolution. By taking into account beneficial effects, we obtain a non-trivial multivariate generalization of their single-type branching process model. Perturbative techniques allows us to obtain analytical asymptotic expressions for the main global parameters of the model, which lead to the following rigorous results: (i) a new criterion for "no sure extinction", (ii) a generalization and proof, for this particular class of models, of the lethal mutagenesis criterion proposed by Bull et al. (J. Virol. 18:2930-2939, 2007), (iii) a new proposal for the notion of relaxation time with a quantitative prescription for its evaluation, (iv) the quantitative description of the evolution of the expected values in four distinct "stages": extinction threshold, lethal mutagenesis, stationary "equilibrium", and transient. Finally, based on these quantitative results, we are able to draw some qualitative conclusions.

Rapid DNA-protein cross-linking and strand scission by an abasic site in a nucleosome core particle.

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.

Onset model of mutually catalytic self-replicative systems formed by an assembly of polynucleotides.

Self-replicability is a unique attribute observed in all living organisms, and the question of how the life was physically initiated could be equivalent to the question of how self-replicating informative polymers were formed in the abiotic material world. It has been suggested that the present DNA and proteins world was preceded by an RNA world in which genetic information of RNA molecules was replicated by the mutual catalytic function of RNA molecules. However, the important question of how the transition occurred from a material world to the very early pre-RNA world remains unsolved both experimentally and theoretically. We present an onset model of mutually catalytic self-replicative systems formed in an assembly of polynucleotides. A quantitative expression of the critical condition for the onset of growing fluctuation towards self-replication in this model is obtained by analytical and numerical calculations.

Detection of single influenza viral RNA in cells using a polymeric sequence probe.

A polynucleotide probe, call a polymeric sequence probe (PSP), was used to detect influenza A (Influenza A/WSN/33) NA (Neuraminidase) viral RNA in Madin-Darby canine kidney (MDCK) cells. The PSP is a single-stranded DNA molecule with ~2,000 tandem repeat fluorescence binding sites and target binding sites that can bind with multiple fluorescence complementary oligos and target viral RNA using a fluorescence in situ hybridization (FISH) process. A single viral RNA labeled by PSP can be directly observed in MDCK cells. The simple FISH protocol enables the observation and quantitative analysis of the infectious process and drug effects with ultrahigh sensitivity and spatial resolution.

Structural basis of the RNase H1 activity on stereo regular borano phosphonate DNA/RNA hybrids.

Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN's are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH(3) modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a R(P) or S(P) BH(3) modification by nuclear magnetic resonance (NMR) spectroscopy. T(M) studies show that the stabilities of R(P) and S(P) modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total R(x) values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the S(P) modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the S(P) BH(3) group in the major groove while the R(P) BH(3) group points away from the DNA. However, this creates a potential clash of the R(P) BH(3) groups with important RNase H1 residues in a complex, while the S(P) BH(3) groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully R(P) BH(3) DNA/RNA hybrid would not be a substrate for RNase H1.

Rapid construction and characterization of synthetic antibody libraries without DNA amplification.

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.

Transcription-coupled repair and mismatch repair contribute towards preserving genome integrity at mononucleotide repeat tracts.

The mechanisms that underpin how insertions or deletions (indels) become fixed in DNA have primarily been ascribed to replication-related and/or double-strand break (DSB)-related processes. Here, we introduce a method to evaluate indels, orientating them relative to gene transcription. In so doing, we reveal a number of surprising findings: First, there is a transcriptional strand asymmetry in the distribution of mononucleotide repeat tracts in the reference human genome. Second, there is a strong transcriptional strand asymmetry of indels across 2,575 whole genome sequenced human cancers. We suggest that this is due to the activity of transcription-coupled nucleotide excision repair (TC-NER). Furthermore, TC-NER interacts with mismatch repair (MMR) under physiological conditions to produce strand bias. Finally, we show how insertions and deletions differ in their dependencies on these repair pathways. Our analytical approach reveals insights into the contribution of DNA repair towards indel mutagenesis in human cells.

Supramolecular assemblies of zwitterionic nanoliposome-polynucleotide complexes as gene transfer vectors: Nanolipoplex formulation and in vitro characterisation.

Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg(2+) on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg(2+) ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.

Site-selective blocking of PCR by a caged nucleotide leading to direct creation of desired sticky ends in the products.

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

Effects of the interaction between beta-carboline-3-carboxylic acid N-methylamide and polynucleotides on singlet oxygen quantum yield and DNA oxidative damage.

The complexation of beta-carboline-3-carboxylic acid N-methylamide (betaCMAM) with the sodium salts of the nucleotides polyadenylic (Poly A), polycytidylic (Poly C), polyguanylic (Poly G), polythymidylic (Poly T) and polyuridylic (Poly U) acids, and with double stranded (dsDNA) and single stranded deoxyribonucleic acids (ssDNA) was studied at pH 4, 6 and 9. Predominant 1:1 complex formation is indicated from Job plots. Association constants were determined using the Benesi-Hildebrand equation. BetaCMAM-sensitized singlet oxygen quantum yields were determined at pH 4, 6 and 9, and the effects on this of adding oligonucleotides, dsDNA and ssDNA were studied at the three pH values. With dsDNA, the effect on betaCMAM triplet state formation was also determined through triplet-triplet transient absorption spectra. To evaluate possible oxidative damage of DNA following singlet oxygen betaCMAM photosensitization, we used thiobarbituric acid-reactivity assays and electrophoretic separation of DNA assays. The results showed no oxidative damage at the level of DNA degradation or strand break.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples.

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5' region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).