Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA

Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are aller-gen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 ho-mo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH enzymology and molecular biology research projects.


Introduction
Glutamate dehydrogenase (GDH EC 1.4.1.2) is a multi-subunit enzyme that polymerizes ribo-nucleoside triphosphates to produce RNA independent of any template [1] but dependent on the enzyme's binomial hexameric subunit composition, a property that is controlled by metabolic environments [2] [3]. Therefore, RNA synthesized by GDH is not genetic. Plant GDH synthesizes large quantities of RNA [4]. The same sequence and population of RNA are synthesized irrespective of the reducing or oxidizing reaction conditions. GDH is functional in all organisms. The metabolic nature of GDH-synthesized RNA provides the opportunity to study the chemical contrasts between genetic code-based and nongenetic code-based RNAs for the differentiation of their biological functions.
They regulate total RNA abundance [5]. GDH-synthesize RNAs have been reverse-transcribed to cDNA and sequenced, leading to large scale preparation of the silencing oligonucleotides, which are routinely applied as tools for monitoring mRNA concentrations, and metabolic networks that coordinate the biochemical pathways [4]. GDH isoenzymes have been induced in whole plants to synthesize selected sets of RNA that knocked out target mRNA populations thereby producing plant metabolic variants that are specialized in the accumulation of many metabolites of dietary importance. Specifically, allergen-free low linoleic acid peanuts [6], fatty acid-enriched peanuts [3], essential amino acid-enriched peanuts [7], ultra-high nutraceutical resveratrol contents of peanut seeds [8], shikimic acid-enriched Phyla dulcis plants [9]; and doubling of biomass yield [4] are some of the biotechnological milestones achieved by unleashing the mRNA degradation activity of GDH-synthesized RNA. The mechanisms of RNA enzyme, and transcript silencing are known [10] [11] [12], but the chemistry by which GDH-synthesized RNA (nongenetic code-based RNA) degrades total RNA (genetic code-based RNA) has not been discussed [5]. RNA-RNA interactions have been studied in detail [13] [14] [15], but the homologous alignment interactions in solutions, between nongenetic code RNA and genetic code-based RNA have not been studied. This paper opens a conversation on the in vitro reactions between GDH-synthesized RNA and total RNA moieties.
Following the biotechnological applications of GDH-synthesized RNA as enzyme, there is need to characterize their chemical properties as compared with genetic code-based RNA. There are many contrasts in the chemistry of coding  [20]. Both coding and noncoding RNAs are however genetic. It is important that genetic code-based and nongenetic code-based RNAs are compared so as to begin to unravel the constraint and freedom imposed on metabolic regulation (siRNA silencing, transcription, replication, translation, genome structure etc.) by genetic code. Methods for investigating RNA generalized molecular properties revolve around the computation for G+C-and A+T-compositions and nearest neighbor base stacking interactions after RNA has been converted to cDNA [21] including algorithmic analyses for probable intramolecular binding sites [13]. Although G+C contents have been applied repeatedly in the description of nucleic acid structure [16] [17] [18] [20], the biochemical basis of the differential distribution of G+C in genomes has not been discussed. Other structural properties of nucleic acids are controlled by hydrogen bonding-related stabilization, melting and annealing temperatures, paramagnetic and electrostatic instability, interactions with protein enzymes and ligands [22] [23] [24]. Most of the properties including the G+C contents could be deduced by subjecting the cDNA to polymerase chain reaction (PCR) amplification, one of the experimental approaches presented hereunder. The structural differences between GDH-synthesized RNA and genetic code-based RNA illuminate the catalytic properties of GDH-synthesized RNA.

Treatment of Experimental Organism
Peanut (Arachis hypogeae Floor Runner cv.) seeds were sterilized in 5% alcohol solution for 10 min, rinsed with deionized water, and planted on moistened filter paper in five replicate petri dishes.

Total RNA
Total RNA was extracted from peanut seedlings using the acidic phenol/chloroform

Purification of GDH Isoenzymes
GDH was purified from the N+N+N+K+S+S-treated peanut seedlings (20 g) by homogenization at 4˚C with 100 mL of buffer [28]

Structural and Functional Characterization of RNAs
To assign putative functions to the RNAs synthesized by GDH (RNA enzyme), their cDNA sequences were used as queries to search the NCBI nucleotide-nucleotide (excluding ESTs) BLAST (blastn) for peanut taxid 3818 database (Arachis hypogaea) [30]. Similarly, to identify the fragments of the cloned

G+C Contents and Amplification Uniformity
Single product bands resulted from the PCR amplification of the insert cDNA of GDH-polymerized RNA (Figure 1), whereas multiple product bands resulted from the amplification of vector insert DNA (genetic code-based DNA), despite that the Tm applied in the "touch-down" PCR was stringent (Table 1), and the PCR protocol was conducted at the same time, with same cocktail of reagents, under same conditions. Therefore, there was amplification stability and uniformity for the cDNAs of GDH-synthesized RNA and amplification instability for the genetic code-based DNA. "Touchdown" PCR is recommended for circumventing incomplete priming [32] [33] [34], and for increased specificity and sensitivity in PCR amplification [35], but many of the recombinant plasmids ( Figure   1    The cDNAs of GDH-synthesized RNA varied their G+C contents broadly covering a range from 35% to 59%, whereas the vector coding DNA fragments varied their G+C contents for a narrower range from 50% to 60% (Table 1).
Therefore, RNA synthesized by GDH is a modified RNA, its cDNA exhibiting chemical properties different from genetic code-based DNA.
The high G+C contents of the vector DNA ( The nearest neighbor interaction is a major factor that affects the stability of nucleic acid, the A.T pairing being always destabilizing [21]. The nearest neighbor effects increased the Tm values to the same extent for both the cDNAs of GDH-synthesized RNA and genetic code-based DNA inserts ( Table 1) It has been suggested that the RNA synthesized by GDH might function in the regulation of mRNA abundance through homologous sequence-mediated RNA interference activity [4] [6] [7]. In describing the features which are correlated with silencing efficiency, Chan et al. [16] identified the importance of low G+C-contents of the siRNA, suggesting that siRNA G+C-content negatively correlated with RNAi efficiency. Liu et al. [19]  in the mRNA for the binding of the GDH-synthesized RNA are also A+U-rich.
Conversely, the higher G+C-contents of genetic code-based DNA (Table 1) could explain some of their inefficient siRNA activities [41]. The target sequences in the mRNA for the binding of the genetic code-based siRNA are also  [9]. Also, mammalian GDH synthesizes RNA.
The differences between the G+C-contents of genetic code-based DNA and nongenetic code-based DNA (Table 1), and in the amplification uniformity of nongenetic code-based DNA ( Figure 1) imply that genetic code-based DNA possess paramagnetic and electrostatic properties [22] [23] [24] that are different from those of nongenetic code-based DNA. Therefore, when GDH-synthesized RNA (the RNA enzyme) aligns in the correct orientation and interacts with its homologous mRNA (genetic code-based RNA) during silencing biochemical reaction, the force of the collusion between the different paramagnetic-dielectric RNA molecules could be so considerable that the resultant energy could liquefy total RNA, the lesser stable of the two types of RNA ( Figure 2).

Reaction between Genetic Code-Based RNA and Nongenetic
Code-Based RNA The reactions between total RNA and the RNA synthesized by GDH resulted to total RNA degradation ( Figure 2). Digital pixelated comparison of the extents of reactions in lanes 1 ( Figure 2: reaction between total RNA of control peanut and the RNA synthesized by the GDH of N+N+N+K+S+S-treated peanut), 2 (total RNA of control peanut, after thermal cycling), 4 (RNA synthesized by the GDH of N+N+N+K+S+S-treated peanut, after thermal cycling), 6 (RNA synthesized by the GDH of N+N+N+K+S+S-treated peanut, before thermal cycling), and 8 (total RNA of control peanut, before thermal cycling) showed that new molecular weight degraded bands lower than the 5S RNA band appeared in lane 1. Comparison of lanes 3 ( Figure 2: reaction between total RNA of N+P+K+K+K-treated peanut and RNA synthesized by the GDH of N+N+N+K+S+S-treated peanut), 2 (total RNA of control peanut, after thermo-cycling), 4 (RNA synthesized by GDH of N+N+N+K+S+S-treated peanut, after thermal cycling), 6 (RNA synthesized by GDH of N+N +N+K+S+S-treated peanut, before thermal cycling), and 8 (total RNA of untreated control peanut, before thermal cycling) also showed the emergence of new degraded RNA bands between the 16S and 5S rRNA bands that were absent from the GDH-synthesized RNA and total RNA in lane 3.   (lanes 2, 4, 6, and 8).
Again, reaction 7 ( Figure 2: lane 7) between the total RNA of N+P+P+P-treated peanut and the RNA synthesized by the GDH of N+N+N+K+S+S-treated peanut displayed a vivid loss of RNA bands compared with the controls (lanes 2, 4, 6, and 8).
The reactions depicted in lanes 1, 3, 5, and 7 in the absence of protein enzymes suggested that the GDH-synthesized RNA acted as enzyme to degrade transcripts that shared sequence homologies with it because comparison of lanes Meyer [14], DiChiacchio et al. [13] applied computational methods to uncover possible interactions between two RNA molecules. Reactions ( Figure 2) were thermo-cycled between 5˚C and 37˚C to increase the rate of homologous sequence alignment, total RNA degradation rate, and to continuously disaggregate the reaction products from the surfaces of the RNA enzyme. We seeded the reactions (Figure 2) by addition of ribo-nucleoside triphosphates to the reaction mix in order to displace the position of equilibrium further to the right.
The results ( Figure 2) were obtained by cross-over reaction between total RNA from a treated peanut and the RNA synthesized by the GDH from a differently treated peanut. When the total RNA and the GDH-synthesized RNA were from the same treatment of peanut, there were no reactions (results not shown). This suggested that the total RNA population in peanut is in steady state relationship with the mechanisms that regulate the synthesis of RNA by the prevalent GDH isoenzymes. This is the biochemical basis of the production of plant metabolic variants (phenotypes) at will, via induction of GDH synthesis of RNA [4] [7] [8] [9] [26].
Again, the molecular responses of the different total RNAs of peanut to the same GDH-synthesized RNA (lanes 1, 3, 5, and 7) were different, further suggesting that each mRNA profile of the total RNAs was for a specific metabolic phenotype/variant [4]. Therefore, the cross-over reactions (Figure 2) were the in vitro demonstration of the in vivo degradation of transcripts by the GDH-synthesized RNA. It could be, but without knowing it, that the biochemical mechanism underlying the ex-

Biochemical Synthesis of G+C and A+U Contents of RNA
In vitro demonstration of the function of GDH-synthesized RNA as enzyme begets a conversation on the structure of the RNA (Table 2 and Table 3). The RNA oligonucleotides synthesized by each GDH hexameric isoenzyme illuminated the chemical frequency of G+C contents because instead of comparing the isoelectric point (pI) values of the polypeptides, GDH biological function is visualized in terms of nucleic acid sequence repeats and homologies ( Table 2) similar to genetic code sequence repeats. GDH has a non-allelic gene structure consisting of three different subunit polypeptides [1]. The gene (GDH 1 ) encoding the more acidic subunits (a, and α) is heterozygous, and co-dominant; and the gene (GDH 2 ) encoding the less acidic subunit (β) is homozygous. The binomial distribution pattern of the 28 hexameric isoenzymes [45] is a protein population array that displays the subunit relationships on the native polyacrylamide gel landscape. The purification of active GDH isoenzymes from the slab of native acrylamide gel was made possible by the subzero electrophoretic fractionation processes that preserved the structural integrity and catalytic function of each hexameric isomer. Systematic pair-wise sequence homology comparisons [31] showed that the RNA (#19) synthesized by GDH β6 homo-hexameric isoenzyme did not share sequence match with the RNA (#18) synthesized by GDH a6 homo-hexameric isoenzyme, and did not share sequence match with the RNA (#17) synthesized by GDH α6 homo-hexameric isoenzyme ( Table 2 and Table 3). This is surprising because the three subunit polypeptides (a, α, and β) of the enzyme share common antigenic properties [26]. However, the RNA (#18) synthesized by GDH a6 homo-hexamer shared three-fold homology matches with the RNA (#17) synthesized by GDH α6 homo-hexameric isoenzyme.
Global homology comparisons of the GDH-synthesized RNAs ( Table 2) of N+N+N+K+S+S-treated peanut showed that most were synthesized by hexameric isoenzymes that consisted of mixed ratios of a-subunit and α-subunit polypeptides; and less frequency of RNAs synthesized by mixed isoenzymes that consisted of the three subunit polypeptides. The RNAs synthesized by nucleotide-treated peanuts were more complicated in their primary structure because almost all of the 28 hexameric isoenzymes of GDH were induced [1]. Therefore, the repeated plus/plus and plus/minus sequence codes (alignments) among the GDH-synthesized oligonucleotide RNAs (

Functional Structure of the GDH-Synthesized RNA
Possibility that the repeated plus/plus and plus/minus sequence matches among the GDH-synthesized oligonucleotide RNAs (Table 2) could resemble nucleic acid genetic codes encouraged a discussion on the functional relationship between genetic codes and the repeats in the GDH-synthesized RNA. The cDNAs of GDH-synthesized RNA were inserted into plasmids and sequenced ( Table 3). The cDNA insert of plasmid 1 (Figure 1) shared one plus/minus sequence match with the mRNA encoding oxalate oxidase of peanut ( Table 2). The cDNA insert of plasmid 2 shared one plus/plus sequence match with the mRNA encoding arachin h5 (profilin). The cDNA insert of plasmid 3 shared one plus/plus  [48]. The cDNA insert of plasmid 7 shared one plus/plus sequence match with the mRNA encoding peanut cultivar fuhua 8 glutamate dehydrogenase 1. The cDNA insert of plasmid 9 shared one plus/minus sequence match with the mRNA encoding peanut strain E2-4-83-12 delta-12 fatty acid desaturase. The cDNA insert of plasmid 10 shared a plus/plus sequence match with the mRNA encoding peanut cultivar fuhua 8 glutamate dehydrogenase 1, the second copy of that silencing RNA synthesized by GDH. The cDNA insert of plasmid 11 shared a plus/plus sequence match with the mRNA encoding peanut cultivar JL24 phenylalanine ammonia-lyase. The cDNA insert of plasmid 13 shared three sequence matches, two of which were plus/plus with +2/+1 frame shifts, and one was minus/minus with −2/−3 frame shifts to the mRNA encoding peanut beta-ketoacyl-ACP synthase ll. This is a complex three-point binding by the GDH-synthesized RNA for wider sequence coverage and total silencing of the mRNA encoding ketoacyl-ACP synthase. Therefore, each insert cDNA of GDH-synthesized RNA (nongenetic code-based RNA) is a unit sequence code for a specific mRNA (genetic code-based RNA).
The DNA insert of plasmid 14 shared sequence match with a section of gnl|uv|U37573.1 phagemid vector pBK-CMV, a component of TOPO TA cloning vector.
The cDNA insert of plasmid 15 shared a plus/plus sequence match with peanut Ara h5 mRNA, being the second copy of that RNA synthesized by GDH. These are evidence that the synthesis of RNA by GDH is reproducible although it is template-independent. The cDNA insert of plasmid 16 shared two plus/minus sequence matches with the mRNA encoding peanut mitogen-activated protein kinase 2 (DQ068453.1); and with the mRNA encoding peanut mitogen-activated protein kinase 3 (EU182580.2). Therefore, the same fragment of RNA synthesized by GDH shared homology with two different mRNAs. This is another example of the complex mechanism, may be involving silencing RNA mass action effect (organic chemical reaction) on mRNA silencing by GDH-synthesized RNA. The cDNA insert of plasmid 17 shared minus/minus sequence match with −1/−3 frame shift to the mRNA encoding Arachis hypogaeae thylene-responsive element binding factor 1 [49]. The cDNA insert of plasmid 18 shared plus/plus sequence match with the mRNA encoding Arachis hypogaea triacylglycerol lipase 1. The cDNA insert of plasmid 19 shared three plus/plus sequence matches with the mRNA encoding peanut mitogen-activated protein kinase 3 (EU182580.2); with the mRNA encoding peanut mitogen-activated protein kinase 2 (DQ068453.1); and with the mRNA encoding peanut mitogen activated protein kinase 1 (DQ068452.1). Plasmid 19 is different from plasmid 16. Therefore, GDH synthesized two different silencing RNA fragments that targeted different sites in the mRNAs encoding the mitogen-activated protein kinases. This  (Table 3) represent the biological codes for recognition of specific mRNA targets in total RNA, the RNA enzyme activity (Figure 2) being the biochemical mechanism for eliminating the undesired mRNAs.

Biotechnological Applications of GDH-Synthesized RNA
In vitro demonstration of the RNA enzyme activity of GDH-synthesized RNA reveals a smart approach to save time, space and effort on basic research experimentations with plants because preliminary surveys of the responses of plant metabolism to the environment could be conducted at reduced scales in growth chambers, greenhouse, and field plots specifically to collect sufficient tissues for total RNA and GDH purifications; the phenotypic responses of the plant to mineral nutrients, biochemical regulators, agro-chemicals and other xenobiotics being conducted and interpreted in biochemical cross-over reactions as in Figure 2. The more severe the degradation of total RNA is, the more suppressed will the biochemical pathways be, and accordingly the more pronounced the phenotypic characteristics of the plant would be. The GDH-synthesized RNA enzyme could be labeled to differentiate its identity from total RNA moieties in the cross-over reactions in Figure 2.
Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts not on base-pairing as in double stranded siRNA, but on the basis of homologous sequence alignment recognition (Figure 2). This nascent biochemical knowledge describing the natural RNA enzyme activity of the ubiquitous GDH synthesized RNA is perhaps of utmost importance in basic enzymology and mo-  [46] however continued to intuitively suggest the existence of an extensive and generalized metabolic network control system at the RNA level but of a somewhat distinct chemical apparatus from the genetic code-based siRNA system [10] [43] [50] [51]. The RNA logic of the generalized chemical silencing apparatus is more vividly illustrated (Figure 2). Therefore, the nongenetic code-based RNA enzyme may provide wider molecular approaches for controlling the population of undesired transcripts in plant systems.
GDH also synthesizes unit oligonucleotides that silence its own encoded mRNAs (Table 3)