ABSTRACT
Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.
ABSTRACT
Intracerebral hemorrhage (ICH) is one of the most lethal types of stroke. Neuroimaging techniques, particularly MRI, have improved the diagnostic accuracy of ICH. The MRI characteristics of the evolving stages of ICH in humans-but not those in dogs-have been described. In this study, we document the temporal MRI characteristics in a canine model of collagenase-induced ICH. Specifically, ICH was induced in 5 healthy beagles by injecting 500 U of bacterial collagenase from Clostridium histolyticum, which was delivered into the parietal lobe over 5 min by using a microinfusion pump. T1- and T2-weighted, fluid-attenuated inversion recovery, gradient-echo (GRE), and diffusion-weighted (DWI) imaging and measurement of the apparent diffusion coefficient (ADC) were performed serially at 6 different time points (before and 12 h, 3 d, 5 d, 10 d and 24 d after hemorrhage) by using a 3-T MR system. The temporal changes of T1 signal intensity (SI) corresponded well with the reported human data. The temporal changes of T2 and GRE sequences, with the exception of T2 and GRE hyperintensities at the early subacute stage, also matched. ADC measurements were high at the early subacute stage, and DWI-SI positively correlated with T2- and GRE-SI from the early subacute stage onward. In conclusion, MRI is an ideal method for characterizing the temporal evolution of parenchymal alterations after ICH in dogs. These data might be useful for differentiating clinical stages of ICH in dogs.