Biology subjects

Oliynyk, R. T.

Publications and source records attributed to Oliynyk, R. T..

9 recordsLinked to original sources

bioRxiv2026

Short single-stranded DNA oligonucleotides enable intracellular transcription of functional RNAs in mammalian cells

Programmable intracellular production of short functional RNAs underlies diverse applications ranging from gene regulation to genome engineering, but is often constrained by cloning workflows, RNA synthesis, or viral-vector delivery. Here, we introduce a single-stranded DNA (ssDNA)-driven intracellular transcription strategy in which short ssDNA templates function as transcriptional substrates when paired with orthogonal bacteriophage RNA polymerases (RNAPs). By embedding phage promoters within short hairpin structures to create a locally double-stranded recognition site, we enable robust RNAP-dependent transcription from ssDNA in mammalian cells. We demonstrate that ssDNA templates can drive production of functional CRISPR guides, supporting adenine base editing at reporter and endogenous loci and enabling CRISPR-based transcriptional activation across distinct human cell lines. These results establish ssDNA templates as compact, synthetically accessible inputs for RNA production in mammalian cells, with broad utility in synthetic biology and genome engineering.

bioengineering↗

bioRxiv2026

DNA cut-ligation cyclization surpasses J-Factor limit by order of magnitude

We demonstrate DNA circularization efficiencies more than threefold greater than those predicted by classical Jacobson-Stockmayer theory for the ligation of linear double-stranded DNA. To quantify deviations from classical expectations, we experimentally calibrated the J-factor by ligating 452 bp DNA fragments bearing pre-cut, purified 4-nt overhangs at the optimal shortest minicircle length and at the highest DNA concentration; this reference value then enabled accurate calculation of expected cyclization efficiencies across the full range of DNA lengths (452-952 bp) and the three concentrations examined. These elevated cyclization efficiencies are enabled by simultaneous restriction cutting and ligation using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions, yielding a 3.4-fold improvement over the classical estimate and achieving 75% cyclization at a high, practically relevant DNA concentration of 120 ng/{micro}l. We also identified a second enzyme, Esp3I, that exceeds the classical expectations by 2.3-fold (though less efficiently than BsaI-HFv2), while BbsI cut-ligation systematically underperformed expectations, providing insight into possible mechanisms underlying the outstanding performance of the first two enzymes. These results reveal a biologically mediated exception that overcomes the long-standing mechanistic expectations of Jacobson- Stockmayer theory and highlight the value of systematically screening enzyme combinations to discover additional systems capable of highly efficient intramolecular ligation.

molecular biology↗

bioRxiv2024

Plasmid2MC: Efficient cell-free recombination of plasmids into high-purity minicircle DNA for use in genome editing applications

AbstractDNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing{Phi} C31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The methods ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.

genetics↗

bioRxiv2022

Persistent Increase in Human-Caused Wolf Mortality Following the Minnesota 2012-2014 Hunting Seasons

By the mid-20th century, wolves were nearly extinct in the lower 48 states, with a small number surviving in northern Minnesota. After wolves were placed on the endangered species list in 1973, the northern Minnesota wolf population increased and stabilized by the early 2000s. A wolf trophy hunt was introduced in 2012-2014 and then halted by a court order in December 2014. The Minnesota Department of Natural Resources collected wolf radiotelemetry data for the years 2004-2019. Statistical analysis showed that wolf mortality remained close to constant from 2004 until the initiation of the hunt, and that mortality doubled with the initiation of the first hunting and trapping season in 2012, remaining at a nearly constant elevated level through 2019. Notably, average annual wolf mortality increased from 21.7% before wolf hunting seasons (10.0% by human causes and 11.7% natural causes) to 43.4% (35.8% by human causes and 7.6% natural causes). The fine-grained statistical trend implies that human-caused mortality increased sharply during the hunting seasons, while natural mortality initially dropped. While gradually diminishing after the discontinuation of the hunt, human-caused mortality remained high, which could indicate a lasting shift in human attitudes and post-hunt changes in wolf behavior and wolf pack composition and size.

ecology↗

bioRxiv2022

Efficient Modification and Preparation of Circular DNA for Expression in Cell Culture

DNA plasmids are an essential tool for delivery and expression of RNAs and proteins in cell culture experiments. The preparation of plasmids typically involves a laborious process of bacterial cloning, validation, and purification. While the expression plasmids can be designed and ordered from the contract manufacturers, the cost may be prohibitive when a large number of plasmids is required. We have developed an efficient fully synthetic method and protocol that enables the production of circularized DNA containing expression elements ready for transfection in as little as 3 hours, thereby eliminating the bacterial cloning steps. The protocol describes how to take a linear double-stranded DNA fragment and efficiently circularize and purify this DNA fragment with minimal hands-on time. As proof of the principle, we applied Circular Vector expressing engineered prime editing guide RNA (epegRNA) in cell culture, and demonstrated matching and even exceeding performance of this method as compared to guides expressed by plasmids. The method is not limited to gene editing and its speed of preparation, low cost, and ease of use will make it a useful tool in a variety of applications requiring the expression of short RNAs and proteins.

genomics↗

bioRxiv2019

Future Preventive Gene Therapy of Polygenic Diseases from a Population Genetics Perspective

With the accumulation of scientific knowledge of the genetic causes of common diseases and continuous advancement of gene-editing technologies, gene therapies to prevent polygenic diseases may soon become possible. This study endeavored to assess population genetics consequences of such therapies. Computer simulations were used to evaluate the heterogeneity in causal alleles for polygenic diseases that could exist among geographically distinct populations. The results show that although heterogeneity would not be easily detectable by epidemiological studies following population admixture, even significant heterogeneity would not impede the outcomes of preventive gene therapies. Preventive gene therapies designed to correct causal alleles to a naturally-occurring neutral state of nucleotides would lower the prevalence of polygenic early- to middle-age-onset diseases in proportion to the decreased population relative risk attributable to the edited alleles. The outcome would manifest differently for late-onset diseases, for which the therapies would result in a delayed disease onset and decreased lifetime risk, however the lifetime risk would increase again with prolonging population life expectancy, which is a likely consequence of such therapies. If gene therapies that prevent heritable diseases were to be applied on a large scale, the decreasing frequency of risk alleles in populations would reduce the disease risk or delay the age of onset, even with a fraction of the population receiving such therapies. With ongoing population admixture, all groups would benefit over generations.

genetics↗

bioRxiv2019

Evaluating the Potential of Younger Cases and Older Controls Cohorts to Improve Discovery Power in Genome-wide Association Studies of Late-onset Diseases

For more than a decade, genome-wide association studies have been making steady progress in discovering the causal gene variants that contribute to late-onset human diseases. Polygenic late-onset diseases in an aging population display the risk allele frequency decrease at older ages, caused by individuals with higher polygenic risk scores becoming ill proportionately earlier and bringing about a change in the distribution of risk alleles between new cases and the as-yet-unaffected population. This phenomenon is most prominent for diseases characterized by high cumulative incidence and high heritability, examples of which include Alzheimers disease, coronary artery disease, cerebral stroke, and type 2 diabetes, while for late-onset diseases with relatively lower prevalence and heritability, exemplified by cancers, the effect is significantly lower. Computer simulations have determined that genome-wide association studies of the late-onset polygenic diseases showing high cumulative incidence together with high initial heritability will benefit from using the youngest possible age-matched cohorts. Moreover, rather than using age-matched cohorts, study cohorts combining the youngest possible cases with the oldest possible controls may significantly improve the discovery power of genome-wide association studies.

genetics↗

bioRxiv2018

Could future gene therapy prevent aging diseases?

BackgroundWithin the next few decades, gene therapy techniques and genetic knowledge may sufficiently advance to support prophylactic gene therapy to prevent polygenic late-onset diseases. A combination of a large number of common low effect gene variants offers the most likely explanation for the heritability of the majority of these diseases, and their risk may be lowered by correcting the effect of a subset of such gene variants.\n\nMethodsComputer simulations quantified the correlation between the aging process, polygenic risk score, and hazard ratio change with age, using as inputs clinical incidence rates and familial heritability, and estimated the outcomes of hypothetical future prophylactic gene therapy on the lifetime risk and age of onset for eight highly prevalent late-onset diseases.\n\nResultsThe simulation results confirmed that gene therapy would be beneficial in both delaying the age of onset and lowering the lifetime risk of the analyzed lateonset diseases. Longer life expectancy is associated with a higher lifetime risk of these diseases, and this tendency, while delayed, will continue after the therapy.\n\nConclusionsWhen the gene therapy as hypothesized here becomes possible, and if the incidences of the treated diseases follow the proportional hazards model with multiplicative genetic architecture composed of a sufficient number of common small effect gene variants, then (a) late-onset diseases with the highest familial heritability will have the largest number of variants available for editing, (b) diseases that currently have the highest lifetime risk, and particularly those with the highest incidence rate continuing into older ages, will prove the most challenging cases in which to lower lifetime risk and delay the age of onset at the populational level, and (c) diseases that are characterized by the lowest lifetime risk will show the strongest and longest-lasting response to such therapy.

genomics↗

bioRxiv2018

Age-related late-onset disease heritability patterns and implications for genome-wide association studies

BackgroundGenome-wide association studies and other computational biology techniques are gradually discovering the causal gene variants that contribute to late-onset human diseases. After more than a decade of genome-wide association study efforts, these can account for only a fraction of the heritability implied by familial studies, the so-called \"missing heritability\" problem.\n\nMethodsComputer simulations of polygenic late-onset diseases in an aging population have quantified the risk allele frequency decrease at older ages caused by individuals with higher polygenic risk scores becoming ill proportionately earlier. This effect is most prominent for diseases characterized by high cumulative incidence and high heritability, examples of which include Alzheimers disease, coronary artery disease, cerebral stroke, and type 2 diabetes.\n\nResultsThe incidence rate for late-onset diseases grows exponentially for decades after early onset ages, guaranteeing that the cohorts used for genome-wide association studies overrepresent older individuals with lower polygenic risk scores, whose disease cases are disproportionately due to environmental causes such as old age itself. This mechanism explains the decline in clinical predictive power with age and the lower discovery power of familial studies of heritability and genome-wide association studies. It also explains the relatively constant-with-age heritability found for late-onset diseases of lower prevalence, exemplified by cancers.\n\nConclusionsFor late-onset polygenic diseases showing high cumulative incidence together with high initial heritability, rather than using relatively old age-matched cohorts, study cohorts combining the youngest possible cases with the oldest possible controls may significantly improve the discovery power of genome-wide association studies.

genetics↗