Application of PCR-based approaches for evaluation of cell-free DNA fragmentation in colorectal cancer

Cell-free DNA (cfDNA) testing is the core of most liquid biopsy assays. In particular, cfDNA fragmentation features could facilitate non-invasive cancer detection due to their interconnection with tumor-specific epigenetic alterations. However, the final cfDNA fragmentation profile in a purified sample is the result of a complex interplay between informative biological and artificial technical factors. In this work, we use ddPCR to study cfDNA lengths in colorectal cancer patients and observe shorter and more variable cfDNA fragments in accessible chromatin loci compared to the densely packed pericentromeric region. We also report a convenient qPCR system suitable for screening cfDNA samples for artificial high molecular weight DNA contamination

колективна монографія

Кримінальний процесуальний кодекс 2012 року: ідеологія та практика правозастосування: колективна монографія / за заг. ред. Ю. П. Аленіна ; відпов. за вип. І. В. Гловюк. - Одеса : Видавничий дім «Гельветика», 2018. - 1148 с

Author's personal copy 5′-flanking sequences can dramatically influence 4.5SH RNA gene transcription by RNA-polymerase III

Received by S.M. Mirkin Keywords: Small RNA 4.5S RNA H 4.5S RNA Internal promoter Mus musculus 4.5SH RNA is a 94 nt small nuclear RNA with an unknown function. Hundreds of its genes are present in the genomes of rodents of six families including Muridae. 4.5SH RNA genes contain an internal RNA-polymerase III promoter consisting of A and B boxes. Here we studied the influence of 5′-flanking sequences on the transcription of a mouse 4.5SH RNA gene. We found that replacement of the upstream sequence can dramatically change the 4.5SH RNA gene transcription efficiency. Various DNA fragments inserted immediately upstream from 4.5SH RNA gene completely inhibited its in vitro transcription, whereas others promoted it. The shortening of the native mouse 5′-flanking sequence of 4.5SH RNA gene to 42 bp resulted in the activation of an additional illegal transcription start site in upstream region. Transcription of the 4.5SH RNA gene with various upstream sequences in transfected HeLa cells revealed the differences between the tests performed in vivo and in vitro: in whole cells, only the construct with 5′-flanking native sequence could be transcribed. Apparently, at least some regions of the native 5′-flanking sequence of 4.5SH RNA genes have been selected during evolution for high transcription activity

Complementarity of end regions increases the lifetime of small RNAs in mammalian cells.

Two RNAs (4.5SH and 4.5SI) with unknown functions share a number of features: short length (about 100 nt), transcription by RNA polymerase III, predominately nuclear localization, the presence in various tissues, and relatively narrow taxonomic distribution (4 and 3 rodent families, respectively). It was reported that 4.5SH RNA turns over rapidly, whereas 4.5SI RNA is stable in the cell, but their lifetimes remained unknown. We showed that 4.5SH is indeed short-lived (t(1/2)~18 min) and 4.5SI is long-lived (t(1/2)~22 h) in Krebs ascites carcinoma cells. The RNA structures specifying rapid or slow decay of different small cellular RNAs remain unstudied. We searched for RNA structural features that determine the short lifetime of 4.5SH in comparison with the long lifetime of 4.5SI RNA. The sequences of genes of 4.5SH and 4.5SI RNAs were altered and human cells (HeLa) were transfected with these genes. The decay rate of the original and altered RNAs was measured. The complementarity of 16-nt end regions of 4.5SI RNA proved to contribute to its stability in cells, whereas the lack of such complementarity in 4.5SH RNA caused its rapid decay. Possible mechanisms of the phenomenon are discussed

Determination of 4.5SH RNA half-life.

<p>(<b>A</b>) Detection of 4.5SH and 4.5SI RNA by Northern hybridization in total RNA isolated after the addition of actinomycin D to KAC cells. 5S rRNA was used as a loading control. (<b>B</b>) Graphs showing the rapid decay of 4.5SH RNA and the stability of 4.5SI RNA. Each graph is based on data from three experiments (error bars, s.d.).</p

Nucleotide sequences of 4.5SH and 4.5SI RNA genes as well as their derivatives.

<p>Nucleotides of 4.5SH and 4.5SI RNA sequences are shown in red and blue, respectively. Green letters correspond to altered nucleotide sequences. Boxes A and B of the pol III promoter are underlined. T_6–7 at the end of constructs are the pol III terminator.</p

The impact of the end stem on the 4.5SI RNA lifetime.

<p>(<b>A</b>) Predicted secondary structures of 4.5SI-derived RNAs. (See the nucleotide sequences of the corresponding constructs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044157#pone-0044157-g003" target="_blank">Fig. 3</a>). Designations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044157#pone-0044157-g005" target="_blank">Fig. 5</a>. (<b>B</b>) Detection of RNA transcribed from the constructs <i>I</i>, <i>J</i>, and <i>K</i> in HeLa cells treated with actinomycin D. 5S rRNA was used as a loading control. (<b>C</b>) Decay kinetics of RNA transcribed from constructs <i>I</i>, <i>J</i>, <i>K</i>, and <i>H</i> (original 4.5SI RNA). Each graph is based on data from three transfection experiments (error bars, s.d.). Average half-lives with standard deviations only for short-living RNAs (<i>I</i> and <i>J</i>) are presented.</p

Determination of 4.5SI RNA half-life.

<p>(<b>A</b>) Northern hybridization of 4.5SI RNA and 5S rRNA in total RNA isolated from KAC cells exposed to actinomycin D for different time periods. (<b>B</b>) Graphs demonstrating the slow decrease of the 4.5SI RNA level in cells following the start of transcription inhibition. Highly stable 5S rRNA was used as a loading control. Error bars, s.d., N = 3.</p

Predicted secondary structures of 4.5SH RNA (<i>A</i>) and its derivatives (<i>B–G</i>) as well as 4.5SI RNA (<i>H</i>).

<p>4.5SH- and 4.5SI-derived nucleotides are marked by red and blue circles. Nucleotides of altered sequences are marked by green circles.</p