Date of Degree
Seth A. Darst
Biochemistry | Molecular Biology
coligos, TS2126 Rnl1, adenylylation, promoterless transcription, small RNA sequencing
Small RNAs (sRNA) such as microRNA (miRNA), small interfering RNA (siRNA), and other types of RNA have been found to play many important biological roles in regulation of different cellular process leading to their possible use as therapeutics. However, one of the biggest hurdles in exploiting these sRNAs in therapeutics has been the difficulty in delivering them safely and stably into human tissues. Many delivery approaches have been undertaken to deliver these sRNA, albeit with many shortcomings in terms of cost, stability, and side effects, necessitating the need for alternative approaches. We are exploring an entirely different approach for conveying sRNA sequence information to cells with minimal cost, and maximum stability by exploiting the cellular RNA polymerase(s) in situ to transcribe small, synthetic, promoterless, single-stranded DNA into sRNA of desired sequence. Our lab previously found that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures could trigger their circumtranscription by human RNA polymerase (RNAP) III in vitro and in human cells. Since they are chemically synthesized, coligos would avoid the problems of viral delivery approach, and maintain sRNA information in a more chemically stable form, i.e. DNA. If transcription based on these coligos can be understood and controlled precisely in terms of their initiation and termination sites, these coligos might provide an alternative means of producing sRNA of desired sequence in human cells. Through the establishment of an in vitro transcription system for coligos using immunoprecipitated RNAP III, we provide here corroborating evidence that RNAP III is the main polymerase responsible for coligo-based transcription. The immunoprecipitated polymerase also enabled us to develop a sequencing protocol for coligo transcripts that will allow the detailed study of coligo transcription with minimal interference from endogenous cellular small RNA sequences. In order to better understand the requirements for productive and precise transcription, structural-activity relationship (SAR) is performed here using more than 20 new coligos. We found that the predicted secondary structure of coligos play important role in promoting productive transcription. Likewise, internal loops and bulges along with their location in relation to the larger terminal ss loop are also important determinants for successful initiation and circumtranscription around the coligo. Circular topology was previously found to be necessary for productive transcription. However, by simply switching the site of circularization from terminal loop region to the helical region, we report here that the linear templates are transcribed nearly as well as their circular counterparts, most probably because of the preservation of intact larger loop-stem junction, where transcription appears to initiate. This observation provides evidence that RNA Pol III can jump a discontinuity, or nick, in its template strand. In parallel, high throughput sequencing analysis of transcripts from 12 different coligos was also performed to deduce the preferred transcription initiation and termination sites in these coligos. Coligo transcription predominantly initiates with rG (52.8%) or rA (33.3%) at stem-loop junction of the larger terminal loop. Transcription initiation sites are homogeneous and confined to a few specific positions in the stem-larger loop junction of coligos compared to the termination sites, which are more heterogeneous. Nevertheless, the majority of termination occurs in the larger terminal loop, indicating the significance of larger terminal loop as a preferred site for transcription initiation and termination. Lessons learned from the structure-activity relation study of different coligos supplemented with their detailed transcripts sequence information provide us with refined information for designing next generation coligos with optimized precision in initiation and to a lesser extent termination sites. Coligos with loop size of 12 bases is desirable if the preferred transcription initiation needs to be in the stem-larger loop junction. Homopolymeric sequences need to be avoided at 3’ end of the stem region to prevent non-template nucleotide addition during transcription initiation slippage. Canonical RNA polymerase III termination signal, if included for triggering termination, should be in the larger loop near 5’ end of coligo stem. Such design, together with the loop size of 12 bases, seems optimal for triggering precise transcription termination in the loop region next to the 5’ end of coligo stem. Above all, with the successful development of high throughput sequencing method for coligo transcripts, we now possess a complete arsenal for exhaustive study of coligo transcription. Finally, we describe a highly efficient method for synthesis of adenylylated DNA sequencing adapters using the coligo circularization enzyme, the thermostable RNA ligase I from bacteriophage TS2126. This alternative adenylylation method will be a very useful and cost-effective method for many RNA biology labs studying high throughput small RNA sequencing.
Lama, Lodoe, "Circular Oligodeoxynucleotides (Coligos) as Chemically Synthesized, Promoter-Independent Small RNA Expression Vectors" (2016). CUNY Academic Works.