Locked nucleic acids based DNA circuits with ultra-low leakage

DNA circuits based on toehold-mediated DNA strand displacement reaction are powerful tools owing to their programmability and predictability.However,performance and practical application of the circuits are greatly restricted by leakage,which refers to the fact that there is no input(invading strand)in the circuit,and the output signal is still generated.Herein,we constructed locked nucleic acids-based DNA circuits with ultra-low leakage.High binding affinity of LNA(locked nucleic acid)-DNA/LNA suppressed the leakage by inhibiting the breathing effect.Based on the strategy,we have built various low-leakage DNA circuits,including translator circuit,catalytic hairpin assembly(CHA)circuit,entropy-driven circuit(EDC),and seesaw circuit.More importantly,our strategy would not affect the desired main reactions:The output signal remained above 85%for all tested circuits,and the signalto-noise ratios were elevated to 148.8-fold at the most.We believe our strategy will greatly promote the development and application of DNA circuits-based DNA nanotechnology.

Loss of silencing activity caused by 5′-terminal modification with D-/L-isonucleotide(isoNA) or locked nucleic acid(LNA) could not be restored by 5′-terminal phosphorylation

In this study,a series of 5′-phosphorylated siRNAs with D-/L-isonucleotide(isoNA)or locked nucleic acid(LNA)incorporated at the 5′-terminus were synthesized.It was found that after incorporating either isoNA or LNA at the 5′-terminus of the antisense strand or sense strand,the silencing activity of modified strands has been inhibited,which cannot be recovered by phosphorylation at the 5′-terminus;however,the silencing activity of unmodified strand to its own target was increased.This work indicates that the isoNA and LNA modification at 5′-terminus can interfere with the strand selection during the RISC assembling process,and the disturbance of the 5′-phosporylation should not be the only viable mechanism.

A designed locked nucleic acid-based nanopore for discriminating ctDNA and its coexisting analogue ncDNA

Circulating tumor DNA(ctDNA),carrying tumor-specific sequence mutations,is a promising biomarker for classification,diagnosis and prognosis of cancers.However,there is still a great challenge in discriminating single-base difference between ctDNA and its coexisting analogue(normal circulating DNA,ncDNA)at a serum sample.A locked nucleic acid(LNA)probe combined with a-HL nanopore sensor was designed,which achieved a high signal-to-background ratio(SBR)of^8.34 × 10^3,as well as a significant discrimination capability(~12.3 times)of single-base diffe rence.The accurate discrimination strategy is label-free,convenient,selective and sensitive,which has great potential in the early diagnosis of diseases and biomedical research fields.

A relative quantitative method to detect OCT4A gene expression by exon-junction primer and locked nucleic acid-modified probe

Objective: OCT4A has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Recent research has shown that OCT4A is also expressed in partial tumor cell lines and tissues. This study is aimed to develop a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for relative quantitative detection of OCT4A mRNA and discrimination from OCT4B, pseudogene, and genomic contaminations. Methods: A locked nucleic acid (LNA)-modified probe was designed to discern the single base difference 352A/C to identify OCT4A mRNA. An exon-junction primer was designed to avoid false positive caused by genomic contaminations. In addition, a house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured in parallel to normalize the differences between samples and operations. Results: Experiments showed that the newly established RT-PCR assay amplified the OCT4A mRNA selectively; OCT4A analogues gave negative signals. Cell lines nTERA-2 and HepG2 showed positive results in OCT4A expression, while for HeLa and 293 cell lines, as well as primary peripheral blood mononuclear cells (PBMCs), OCT4A expression was negative. Additionally, the relative quantity of OCT4A mRNA was calculated by cycle threshold (Ct) method and house keeping gene normalization. Conclusions: This technique proved to be effective for relative quantitation of OCT4A mRNA with high specificity.